Methods for Objective Assessment of Memory, Early Detection of Risk for Alzheimer&#39;s Disease, Matching Individuals With Treatments, Monitoring Response to Treatment, and New Methods of Use for Drugs

ABSTRACT

Disclosed are methods for assessing severity, determining future risk, matching with a drug treatment, and measuring response to treatment, for memory dysfunction, Alzheimer&#39;s disease, and cognitive decline. Also disclosed are new methods of use for drugs and natural compounds repurposed for use in improving memory, as well as for preventing and treating memory disorders, Alzheimer&#39;s disease and cognitive decline. All the above-mentioned methods are computer assisted methods analyzing the expression of panels of genes, clinical measures, and drug databases. A universal approach in everybody, as well as a personalized approaches by gender, and by diagnosis, are disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.62/852,081 filed on May 23, 2019. This application is incorporatedherein by reference in its entirety for all purposes.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under OD007363 awardedby the National Institutes of Health and CX000139 merit award by theVeterans Administration. The government may have rights in theinvention.

BACKGROUND

Alzheimer's disease is a clear and present danger to older adults, andhas a profound socio-economic impact. Existing therapies are limited inefficacy. Early identification of subjects at risk may open the door topreventive approaches. Short-term memory dysfunction is a key earlyfeature of Alzheimer's disease. Psychiatric patients may be at higherrisk for memory dysfunction and subsequent Alzheimer's disease due tothe negative effects of stress and depression on the brain.

Existing drugs have potential utility in other diseases and disorders.Biomarkers can serve as companion diagnostics for clinical trials forthe development of new medications and also for repurposing existingdrugs for other diseases and disorders.

Accordingly, methods are needed for early identification of memorydysfunction and Alzheimer's disease. Additionally, methods are neededfor identifying and repurposing existing drugs and natural compounds foruse as treatments of other disorders and diseases.

SUMMARY

The present disclosure is generally directed at methods for assessingmemory dysfunction and early identification/prediction of risk forfuture memory dysfunction, Alzheimer's disease and cognitive decline,using computer assisted methods that derive scores based on biomarkerdata, in some instances blood biomarker data. Further, the presentdisclosure relates to methods for matching individuals with drugs toreduce the risk of and mitigate memory dysfunction, Alzheimer's diseaseand cognitive decline, and methods for monitoring response to treatment.Finally, the invention relates to new methods of use for candidate drugsand natural compounds repurposed for treating memory dysfunction,Alzheimer's disease and cognitive decline. All the above-mentionedmethods may include computer-assisted methods that generate scores basedon analyses of the expression of panels of genes, clinical measures, anddrug databases. A universal approach in everybody, as well as apersonalized approach by gender, and by diagnosis, are disclosed.

In one aspect, the present disclosure is directed to a method foridentifying a biomarker for Alzheimer's disease, the method comprising:obtaining a first biological sample from a subject and administering afirst memory test to the subject; obtaining a second biological samplefrom the subject and administering a second memory test to the subject;identifying a first cohort of subjects by identifying subjects havingabout 20% change in a memory retention characteristic as determined by adifference between the first memory test and the second memory test;identifying candidate biomarkers in the first cohort by identifyingbiomarkers having a change in expression.

In one aspect, the present disclosure is directed to a method to reducethe risk of and mitigate memory dysfunction, Alzheimer's disease, andcognitive decline in a subject in need thereof, the method comprisingadministering a therapy to the subject, the therapy being selected fromthe group consisting of one or more compounds from Tables 5A1-A5, and5B1-B5, and 5C1-C2.

In one aspect, the present disclosure is directed to acomputer-implemented method for assessing a low memory state in asubject, and for assessing risk of future Alzheimer Disease andcognitive decline in a subject, the method comprising: computing a scorebased on RNA level, protein level, DNA methylation, a single nucleotidepolymorphism, a panel of at least one biomarker in one of Table 2, Table4A and Table 4B, and combinations thereof in a sample obtained from asubject; computing a score based on a reference expression level of thepanel of biomarkers; and identifying a difference between the score inthe sample obtained from the subject and the score in the referencesample, wherein the difference in the score in the sample obtained fromthe subject and the score in the reference sample indicates a risk for alow memory state in the subject. In other aspects, the presentdisclosure is directed to a method for assessing and mitigating memorydysfunction, Alzheimer's disease, and cognitive decline in a subject inneed thereof, comprising determining an expression level of a panel ofbiomarkers listed in Table 2, Table 4, or Table 5 in a sample, whereinthe expression level of the biomarkers in the sample is differentrelative to a reference expression level, identifying the subjectcurrently having or at risk of having in the future memory dysfunction,Alzheimer's disease, and cognitive decline based on a biomarker panelscore relative to a biomarker panel score of a reference; andadministering to the subject a therapy being selected based on the scorefrom the group consisting of one or more compounds from Tables 5A1-A5,and 5B1-B5, and 5C1-C2.

In some aspects, of the disclosed methods, the therapy is lithium, anantidepressant, pioglitazone, sulfadimidine, SB-203580, mesalazine,metamizole, levonorgestrel, meglumine, lymecycline, rimexolone,ketanserin, quipazine, cisapride, proparacaine, tenoxicam, bexarotene,an omega-3 fatty acid, salsolidine, ginkgolide A, icariin,docosahexaenoic acid, or combinations thereof.

In some aspects, the sample comprises a peripheral tissue, blood,saliva, cerebrospinal fluid (CSF), serum, urine, or stool.

In other aspects, the present disclosure is directed to a compositioncomprising one or more compounds from Tables 5A1-A5, and 5B1-B5, and5C1-C2 for use in a method for treating memory dysfunction, Alzheimer'sdisease, and cognitive decline.

In some aspects, the compound comprises lithium, an antidepressant,pioglitazone, sulfadimidine, SB-203580, mesalazine, metamizole,levonorgestrel, meglumine, lymecycline, rimexolone, ketanserin,quipazine, cisapride, proparacaine, tenoxicam, bexarotene, an omega-3fatty acid, salsolidine, ginkgolide A, icariin, docosahexaenoic acid, orcombinations thereof. In some aspects, the compound comprises one ormore of the compounds from Tables 5A1-A5, and 5B1-B5, and 5C1-C2.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are illustrations depicting the methods described in thepresent disclosure. FIG. 1A depicts the cohorts used in study, depictingflow of discovery, prioritization, and testing of biomarkers. FIG. 1Bdepicts the differential gene expression in the discovery cohort-numberof genes identified with differential expression (DE) and absent-present(AP) methods with an internal score of 2 and above. In FIG. 1C, thepyramid on the left depicts the number of discovery step probesets,identified based on their score for tracking memory, with a maximum ofinternal points of 6 (33% (2 pt), 50% (4 pt) and 80% (6 pt)), and thepyramid on the right depicts prioritization with CFG for prior evidenceof involvement in AD.

FIG. 2 is a schematic illustrating the interaction networks for topcandidate biomarkers (n=111 top genes, 136 probe sets).

FIGS. 3A and 3B are graphs depicting the best single biomarkers forpredictors of state (low memory retention state) (FIG. 3A) and trait(future neuropsychosis) (FIG. 3B). Bold—top CFG scoring biomarkers onthe list (CFG≥12, n=21 probe sets). Bar graph shows best predictivebiomarkers in each group. *Nominally significant p<0.05. Tableunderneath the figures displays the actual number of biomarkers for eachgroup whose ROC AUC p-values (FIG. 3A) and Cox Regression Odds Ratiop-values (FIG. 3B) are at least nominally significant. Some femalediagnostic groups were not shown in the graph as they did not havesubjects to be tested or any significant biomarkers. Cross-sectional wasbased on levels at one visit. Longitudinal was based on levels atmultiple visits (integrates levels at most recent visit, maximum levels,slope into most recent visit, and maximum slope). Dividing linesrepresent the cutoffs for a test performing at chance levels (white),and at the same level as the best biomarkers for all subjects incross-sectional (gray) and longitudinal (black) based predictions. Allbiomarkers performed better than chance. Biomarkers performed betterwhen personalized by gender and diagnosis.

FIGS. 4A and 4B are graphs depicting RHEB as a possible personalizedbiomarker predictor for risk of future AD in Males with Schizophrenia.Subject Phchp098 was a male with schizophrenia (SZ) tested in 2009. Hewas first diagnosed with paranoid schizophrenia in 1977. In 2016, he wasalso diagnosed by neuropsychological testing with ADRD and impaireddecision-making capacity. At that time, he was 66 years old. Subject wasthe only one so far with an ADRD diagnosis in the independentreplication follow-up cohort. RHEB levels were Z-scored by gender anddiagnosis. Subject Phchp098 had the highest levels of RHEB in testingfrom all the subjects with future neuropsychological testing (FIG. 4A),and in fact the highest level of RHEB from all the 111 subjects in thatcohort (FIG. 4B).

FIG. 5 is a schematic illustrating the pharmacogenomics of the topbiomarkers modulated by existing drugs.

FIG. 6 is a schematic diagram depicting the matching of patients todrugs, the pharmacogenomics.

DETAILED DESCRIPTION

Disclosed are methods for identifying biomarkers for memory dysfunctionand early identification of Alzheimer's disease. Also disclosed aremethods using biomarker expression levels for identifying and treatingone or more populations or subpopulations for reducing risk of andmitigating memory dysfunction, Alzheimer's disease, and cognitivedecline. Further, the present disclosure relates to methods foridentifying candidate drugs and natural compounds repurposed fortreating memory dysfunction, Alzheimer's disease and cognitive decline.The methods are useful for early detection of Alzheimer's disease insubjects and identifying existing drugs and natural compounds that canbe repurposed for treating subjects for memory dysfunction, Alzheimer'sdisease and cognitive decline.

In one aspect, the present disclosure is directed to a method foridentifying a one or more biomarker(s) for Alzheimer's disease, themethod comprising: obtaining a first biological sample from a subjectand administering a first memory test to the subject; obtaining a secondbiological sample from the subject and administering a second memorytest to the subject; identifying a first cohort of subjects byidentifying subjects having about 20% change in a memory retentioncharacteristic as determined by a difference between the first memorytest and the second memory test; identifying candidate biomarker(s) inthe first cohort by identifying biomarkers having a change inexpression.

The method can further include prioritizing the candidate biomarkers byidentifying candidate biomarkers known to be associated with Alzheimer'sdisease.

A suitable memory test is Hopkins Verbal Learning Test-Revised (HVLT-R).Suitable subjects include those having a psychiatric disorder. Suitablesubjects can be male subjects and female subjects.

As used herein, “sample” or “biological sample” refers to the samplefrom which biomarkers are measured. In some embodiments, the sample isblood. In some embodiments, the sample can be saliva, cerebrospinalfluid (CSF), serum, urine, stool, and/or another bodily fluid. In someembodiments, the sample is a peripheral tissue.

As used herein, “expression level of a biomarker” refers to the processby which a gene product is synthesized from a gene encoding thebiomarker as known by those skilled in the art. The gene product can be,for example, RNA (ribonucleic acid) and protein. Expression level can bequantitatively measured by methods known by those skilled in the artsuch as, for example, northern blotting, amplification, polymerase chainreaction, microarray analysis, tag-based technologies (e.g., serialanalysis of gene expression and next generation sequencing such as wholetranscriptome shotgun sequencing or RNA-Seq), Western blotting, enzymelinked immunosorbent assay (ELISA), and combinations thereof. In someembodiments, the biomarker is a polymorphic biomarker profile. In someembodiments, the polymorphic biomarker profile includes one or moresingle nucleotide polymorphisms (SNPs), one or more restriction fragmentlength polymorphisms (RFLPs), one or more short tandem repeats (STRs),one or more variable number of tandem repeats (VNTRs), one or morehypervariable regions, one or more minisatellites, one or moredinucleotide repeats, one or more trinucleotide repeats, one or moretetranucleotide repeats, one or more simple sequence repeats, or one ormore insertion elements. In some embodiments, the methods furtherinclude establishing a profile of biomarkers.

As used herein, “a reference expression level of a biomarker” refers tothe expression level of a biomarker established for a subject with noknown memory dysfunction, Alzheimer's disease and cognitive decline,expression level of a biomarker in a normal/healthy subject with noknown memory dysfunction, Alzheimer's disease and cognitive decline asdetermined by one skilled in the art using established methods asdescribed herein, and/or a known expression level of a biomarkerobtained from literature. The reference expression level of thebiomarker can further refer to the expression level of the biomarkerestablished for a high risk subject for memory dysfunction, Alzheimer'sdisease and cognitive decline, including a population of high risksubjects. The reference expression level of the biomarker can also referto the expression level of the biomarker established for a low riskmemory dysfunction, Alzheimer's disease and cognitive decline subject,including a population of low risk subjects. The reference expressionlevel of the biomarker can also refer to the expression level of thebiomarker established for any combination of subjects such as a subjectwith no known memory dysfunction, Alzheimer's disease and cognitivedecline, expression level of the biomarker in a normal/healthy subjectwith no known memory dysfunction, Alzheimer's disease and cognitivedecline, expression level of the biomarker for a subject who has nomemory dysfunction, Alzheimer's disease and cognitive decline at thetime the sample is obtained from the subject, but who later exhibitsmemory dysfunction, Alzheimer's disease and cognitive decline. Forexample, depending on the biomarker(s) selected, the difference in theexpression level of the biomarker(s) can indicate an increased (greater)risk that a subject will develop symptoms consistent with memorydysfunction, Alzheimer's disease and cognitive decline. Conversely,depending on the biomarker(s) selected, the difference in the expressionlevel of the biomarker(s) can indicate a decreased (lower) risk that asubject will develop symptoms with or memory dysfunction, Alzheimer'sdisease and cognitive decline.

In some embodiments, the methods can further include genotyping thesubject. The genotyping can be performed by methods such as sequencing,nucleic acid array and PCR. The nucleic acid can be double-stranded DNA,single-stranded DNA, single-stranded DNA hairpins, DNA/RNA hybrids, RNA,RNA hairpins and cDNA. The presence or absence of the one or morenucleic acids can be determined by sequencing, nucleic acid array andPCR. Suitable nucleic acid arrays include DNA arrays such as, forexample polymorphism arrays. Suitable polymorphism arrays include SNParrays, for example.

In one aspect, the present disclosure is directed to a method foridentifying a subject suspected of having Alzheimer's disease, themethod comprising: obtaining a first biological sample from a subject;obtaining a second biological sample from the subject; and identifyingthe subject by identifying a change in expression of at least one ofRAB7A, NPC2, TGFB1, GAP43, ARSB, PERI, GUSB, MAPT, FCGR1A, UBE2L3, NKTR,RHEB, PTGS2, RGS10, ITPKB, KIDINS220, GSK3B, SERTAD3, APOE, UBE2I,FOXO3, THRA, IGF1, NPTX2, GSTM3, BACE1, PSEN1, GFAP, TREM2, NOCT,CEP350, PPP2R2B, NRP2, CTSS, VEGFA, and combinations thereof.

The method can further include administering a memory test to thesubject when the first biological sample is obtained from the subjectand administering the memory test to the subject when the secondbiological sample is obtained from the subject; and determining a changein a memory retention characteristic as determined by a differencebetween the first memory test and the second memory test. Suitably, thememory test is Hopkins Verbal Learning Test-Revised (HVLT-R). The HVLT-Rcan be used to determine a ‘Low Memory Retention’, which as used herein,can also be called ‘Low Memory State’ or ‘Low Memory Retention state’ or‘Memory Retention measure.’ Suitably, the subject can have about 20%change in a memory retention characteristic as determined by adifference between the first memory test and the second memory test.

Suitable subjects include those having a psychiatric disorder. Suitablesubjects can be male subjects and female subjects.

Suitable subjects include subjects over 21 years old.

In one aspect, the present disclosure is directed to a method ofprophylactically treating a subject for Alzheimer's Disease, the methodcomprising: obtaining a first biological sample from a subject;obtaining a second biological sample from the subject; and identifying achange in expression of at least one of RAB7A, NPC2, TGFB1, GAP43, ARSB,PERI, GUSB, MAPT, FCGR1A, UBE2L3, NKTR, RHEB, PTGS2, RGS10, ITPKB,KIDINS220, GSK3B, SERTAD3, APOE, UBE2I, FOXO3, THRA, IGF1, NPTX2, GSTM3,BACE1, PSEN1, GFAP, TREM2, NOCT, CEP350, PPP2R2B, NRP2, CTSS, VEGFA, andcombinations thereof; identifying a difference between the expressionlevel of the at least one of RAB7A, NPC2, TGFB1, GAP43, ARSB, PERI,GUSB, MAPT, FCGR1A, UBE2L3, NKTR, RHEB, PTGS2, RGS10, ITPKB, KIDINS220,GSK3B, SERTAD3, APOE, UBE2I, FOXO3, THRA, IGF1, NPTX2, GSTM3, BACE1,PSEN1, GFAP, TREM2, NOCT, CEP350, PPP2R2B, NRP2, CTSS, VEGFA, andcombinations thereof, and a reference expression level of at least oneof RAB7A, NPC2, TGFB1, GAP43, ARSB, PERI, GUSB, MAPT, FCGR1A, UBE2L3,NKTR, RHEB, PTGS2, RGS10, ITPKB, KIDINS220, GSK3B, SERTAD3, APOE, UBE2I,FOXO3, THRA, IGF1, NPTX2, GSTM3, BACE1, PSEN1, GFAP, TREM2, NOCT,CEP350, PPP2R2B, NRP2, CTSS, VEGFA, and combinations thereof; andadministering a therapy to the subject.

Suitable therapies can include a drug, a natural compound, andcombinations thereof. Suitable drugs can include lithium, anantidepressant, pioglitazone, levonorgestrel, and bexarotene, forexample. Suitable natural compounds can include omega-3 fatty acid(e.g., docosahexaenoic acid), salsolidine, ginkgolide A, and icariin,for example.

In one aspect, the present disclosure is directed to a method foridentifying a biomarker (e.g., a blood biomarker) for short-term memorydysfunction, the method comprising: obtaining a first biological samplefrom a subject and administering a first memory test to the subject;obtaining a second biological sample from the subject and administeringa second memory test to the subject; identifying a first cohort ofsubjects by identifying subjects having about 20% change in a memoryretention characteristic as determined by a difference between the firstmemory test and the second memory test; identifying candidate biomarkersin the first cohort by identifying biomarkers having a change inexpression; and prioritizing the candidate biomarkers by identifyingcandidate biomarkers known to be associated with short-term memory.

The can further include prioritizing the candidate biomarkers byidentifying candidate biomarkers known to be associated with short-termmemory.

A suitable memory test is Hopkins Verbal Learning Test-Revised (HVLT-R).

Suitable subjects include those having a psychiatric disorder. Suitablesubjects can be male subjects and female subjects.

In one aspect, the present disclosure is directed to a method foridentifying a drug candidate for repurposing for use in treatingAlzheimer's disease, the method comprising: obtaining a first biologicalsample from a subject and administering a first memory test to thesubject; obtaining a second biological sample from the subject andadministering a second memory test to the subject; identifying a firstcohort of subjects by identifying subjects having about 20% change in amemory retention characteristic as determined by a difference betweenthe first memory test and the second memory test; identifying acandidate biomarker in the first cohort by identifying a biomarkerhaving a change in expression; identifying a drug having an effect onthe biomarker; and identifying the drug as a candidate for treatingAlzheimer's disease.

Suitable drugs include those that reduce the activity of the biomarker.Other suitable drugs include those that increases the activity of thebiomarker.

The biomarker is at least one of RAB7A, NPC2, TGFB1, GAP43, ARSB, PERI,GUSB, MAPT, FCGR1A, UBE2L3, NKTR, RHEB, PTGS2, RGS10, ITPKB, KIDINS220,GSK3B, SERTAD3, APOE, UBE2I, FOXO3, THRA, IGF1, NPTX2, GSTM3, BACE1,PSEN1, GFAP, TREM2, NOCT, CEP350, PPP2R2B, NRP2, CTSS, VEGFA, andcombinations thereof.

In one aspect, the present disclosure is directed to a method foridentifying a subject having or at risk for having cognitive decline,the method comprising: obtaining a first biological sample from asubject and administering a first memory test to the subject; obtaininga second biological sample from the subject and administering a secondmemory test to the subject; identifying a first cohort of subjects byidentifying subjects having about 20% change in a memory retentioncharacteristic as determined by a difference between the first memorytest and the second memory test; identifying candidate biomarkers in thefirst cohort by identifying biomarkers having a change in expression;and prioritizing the candidate biomarkers by identifying candidatebiomarkers known to be associated with cognitive decline.

The method can further include prioritizing the candidate biomarkers byidentifying candidate biomarkers known to be associated with cognitivedecline.

A suitable memory test is Hopkins Verbal Learning Test-Revised (HVLT-R).

In one embodiment, the subject also has a psychiatric disorder.

Suitable subjects are male subjects and female subjects.

The cognitive decline can be cognitive impairment dysfunction, mildcognitive impairment, and dementia.

In one aspect, the present disclosure is directed to a method ofprophylactically treating a subject for cognitive decline, the methodcomprising: obtaining a first biological sample from a subject;obtaining a second biological sample from the subject; and identifying achange in expression of at least one of RAB7A, NPC2, TGFB1, GAP43, ARSB,PERI, GUSB, MAPT, FCGR1A, UBE2L3, NKTR, RHEB, PTGS2, RGS10, ITPKB,KIDINS220, GSK3B, SERTAD3, APOE, UBE2I, FOXO3, THRA, IGF1, NPTX2, GSTM3,BACE1, PSEN1, GFAP, TREM2, NOCT, CEP350, PPP2R2B, NRP2, CTSS, VEGFA, andcombinations thereof; and administering a therapy to the subject.

Suitable therapies include drugs, natural compounds, and combinationsthereof. In one embodiment, the subject can also have a psychiatricdisorder. In s

Suitable subjects are male subjects and female subjects.

The cognitive decline is cognitive impairment dysfunction, mildcognitive impairment, and dementia.

The method can further include obtaining a memory impairment score fromthe subject by administering a memory impairment screening test to thesubject. A suitable memory test is Hopkins Verbal Learning Test-Revised(HVLT-R).

In some embodiments, the method includes converting the Z-scoredexpression value of each biomarker into a numeric score of 1, 0.5 or 0,depending if the biomarker's expression is in the high-risk range,intermediate risk range, or low risk range, based on the referenceexpression values for the particular biomarker. In some instances, thisscore is multiplied by the biomarker's CFE (Convergent FunctionalEvidence) score, which serves as a weight, as not all biomarkers areequally important. See such CFE scores in Table 2. In some instances,the resulting value is then divided by the maximum possible CFE scorefor that particular biomarker, yielding a weighted score. In someinstances, the weighted scores are added for all the biomarkers in thepanel, and divided by the number of markers in the panel. In someinstances, the panel score is multiplied by 100 to generate a valuebetween 0 and 100, which can be compared to a reference score.

In some embodiments, for each biomarker in the panel, a list of existingpsychiatric medications that modulate the expression of the biomarker inthe direction of high memory can be identified bioinformatically. Insome instances, each such medication can be given a score commensuratewith the biomarker score, i.e. 1 or 0.5 or 0. In some instances, such amedication can modulate more than one biomarker. In some intances, anaverage score for each medication can be calculated based on its effectson the biomarkers in the panel, and multiplied that by 100, resulting ina score of 0 to 100 for each medication. In some embodiments,psychiatric medications can be matched to the expression of biomarkersin a particular patient and ranked in order of impact on the panel.

In some embodiments, large drug gene expression databases such asConnectivity Map and NIH LINCS can be interrogated, as related toparticular biomarkers that are positive as high risk in the panel in aparticular patient. In some instances, this can lead to anindividualized drug repurposing, identifying and ranking for fit using ascore. As such, a new method of use for non-psychiatric medications andnutraceuticals can be identified and used in a particular patient toreduce risk and mitigate memory dysfunction, Alzheimer's Disease andcognitive decline.

EXAMPLES Materials and Methods

Two independent cohorts of psychiatric disorders patients, one forDiscovery of candidate biomarkers, and one for Testing of top biomarkers(for predicting memory state, and predicting future positiveneuropsychological testing for cognitive impairment) were used (FIG. 1,Table 1).

TABLE 1 Aggregate demographics. Cohorts used in study. Number Age inyears at of time of lab visit subjects Mean (number (SD) T-test for ageat Cohorts of visits) Gender Diagnosis Ethnicity (Range) time of labvisit Discovery Discovery Cohort 159 Male = 131 (414) BP = 52 (187) EA =107 (347) 50.26 (Within-Subject (with Female = 28 (82) MDD = 23 (64) AA= 47 (135) (8.97) Changes in 496 SZA = 35 (97) Asian = 1 (2) (22-66)Memory visits) SZ = 27 (82) Hispanic = 3 (9) Retention) PTSD = 14 (43)Biracial = 1 (3) MOOD = 5 (14) PSYCH = 3 (9) Testing Independent 127Male = 97 (176) BP = 37 (73) EA = 86 (162) 50.48 Low Memory TestingCohort (238  Female = 30 (62) MDD = 24 (48) AA = 40 (73) (8.2) Retention (n = 68) vs. For Predicting visits) SZA = 27 (48) Asian = 1(3) (23-74) Others (n = 170) State (Low SZ = 23 (42) Low Memory 0.703983Memory PTSD = 12 (20) Retention = 50.9 (10.9) Retention ≤40 MOOD = 2 (5)Others = 50.32 (6.83) at Time of PSYCH = 2 (2 Assessment) Independent 56 Male = 47 (91) BP = 11 (23) EA = 33 (64) 55.6  Future PositiveTesting Cohort For (111  Female = 9 (20) MDD = 13 (26) AA = 23 (47)(5.0)  Neuropsych Predicting Trait visits) SZA = 11 (20) (40-74) Testing(n = 11) vs. (Future Positive SZ = 15 (30) Neuropsych Testing Others (n= 100) Neuropsych PTSD = 5 (10) Positive = 54.2 (6.05) 0.411644 Testingfor MOOD = 1 (2) Others = 55.8 (4.89) Dementia in All Years FollowingAssessment) BP-Bipolar; MDD-Major depressive disorder;SZA-schizoaffective disorder; SZ-schizophrenia, PTSD-post-traumaticstress disorder.

The psychiatric subjects were part of a larger longitudinal cohort ofadults. Subjects were recruited from the patient population at theIndianapolis VA Medical Center. All subjects understood and signedinformed consent forms detailing the research goals, procedure, caveatsand safeguards, per IRB approved protocol. Subjects completed diagnosticassessments by an extensive structured clinical interview —DiagnosticInterview for Genetic Studies, and up to six testing visits, 3-6 monthsapart or whenever a new psychiatric hospitalization occurred. At eachtesting visit, they received a series of rating scales, including aHopkins Verbal Learning Test (HVLT-R, see FIG. 6), and blood was drawn.Whole blood (10 ml) was collected in two RNA-stabilizing PAXgene tubes,labeled with an anonymized ID number, and stored at −80° C. in a lockedfreezer until the time of future processing. Whole-blood RNA wasextracted for microarray gene expression studies from the PAXgene tubes,as detailed below.

For this study, the within-subject longitudinal discovery cohort, fromwhich the biomarker data were derived, consisted of 159 subjects (131males, 28 females) with multiple testing visits (a total of 496), whoeach had at least one 20% change in the Retention measure of HVLT fromone consecutive testing visit to another.

The independent test cohort for predicting state (Low Memory Retention)consisted of 127 subjects (97 males, 30 females), demographicallymatched with the discovery cohort, with one or more testing visits (fora total of 238 visits). Low Memory Retention was defined as a score of≤40 (FIG. 1, Table 1).

The independent test cohort for predicting trait (future positiveneuropsychological testing for cognitive impairment) consisted of 56subjects (47 males, 9 females), demographically matched with thediscovery cohort, with one or more testing visits in our lab (for atotal of 111 visits). Positive neuropsychological testing was defines asa diagnosis of MCI, ADRD (Alzheimer Disorder Related Dementia), or otherdementia upon neuropsychological testing done in a clinical setting,triggered by clinical concerns as part of regular clinical care (FIG. 1,Table 1).

Medications. The subjects in the discovery cohort were all diagnosedwith various psychiatric disorders (see, Table 1), and had variousmedical co-morbidities. Their medications were listed in theirelectronic medical records, and documented at the time of each testingvisit. Medications can have a strong influence on gene expression.However, the discovery of differentially expressed genes was based onwithin-subject analyses, which factor out not only genetic backgroundeffects but also minimizes medication effects, as the subjects rarelyhad major medication changes between visits. Moreover, there was noconsistent pattern of any particular type of medication, as the subjectswere on a wide variety of different medications, including bothpsychiatric and non-psychiatric. Furthermore, the independent validationand testing cohorts' gene expression data was Z-scored by gender anddiagnosis before being combined, to normalize for any such effects.

RNA extraction. Whole blood (2.5-5 ml) was collected into each PaxGenetube by routine venipuncture. PaxGene tubes contain proprietary reagentsfor the stabilization of RNA. RNA was extracted and processed aspreviously described (Niculescu et al., Mol. Psychiatry 2015 20(11):1266-1285; Levey et al., Mol. Psychiatry 2016 21(6): 768-785;Le-Niculescu et al., Mol. Psychiatry 2013 18(12): 1249-1264).

Microarray. Microarray work was carried out as previously described(Niculescu et al., Mol. Psychiatry 2015 20(11): 1266-1285; Levey et al.,2016; Le-Niculescu et al., 2013.

For biomarker discovery, the subject's score from the HVLT-DR Retentionmeasure was assessed at the time of blood collection (FIG. 1). Using a20% change threshold in Retention, differences in gene expressionbetween visits were analyzed, using a powerful within-subject design,then an across-subjects summation (FIG. 1).

Data was analyzed in two ways: an Absent-Present (AP) approach, and adifferential expression (DE) approach. The AP approach may captureturning on and off of genes, and the DE approach may capture gradualchanges in expression. A powerful within-subject design, then anacross-subjects summation score was used for probe sets. Affymetrixmicroarray data was imported as CEL. files into Partek Genomic Suites6.6 software package (Partek Incorporated, St Louis, Mich., USA). Usingonly the perfect match values, a robust multi-array analysis (RMA) bygender and diagnosis, background corrected with quantile normalizationand a median polish probe set summarization of all chips, was performedto obtain the normalized expression levels of all probe sets for eachchip. Then, to establish a list of differentially expressed probe sets awithin-subject analysis was conducted using a fold change in expressionof at least 1.2 between high stress and low stress visits within eachsubject. Probe sets that had a 1.2-fold change were then assigned eithera 1 (increased in high stress) or a −1 (decreased in high stress) ineach comparison. These values were then summed for each probe set acrossall the comparisons and subjects, yielding a range of raw scores. Theprobe sets above the 33.3% of scores received an internal score of 2points, those above 50% received 4 points, and those above 80% received6 points. R scripts were developed to automate and conduct all theselarge dataset analyses in bulk, and checked against human manualscoring.

Gene Symbol for the probe sets were identified using NetAffyx(Affymetrix) for Affymetrix HG-U133 Plus 2.0 GeneChips, followed byGeneCards to confirm the primary gene symbol. In addition, for thoseprobe sets that were not assigned a gene symbol by NetAffyx, GeneAnnotor UCSC were used to obtain gene symbols, followed by GeneCard. Geneswere then scored using the manually curated CFG databases as describedbelow (FIG. 1).

For prioritization using Convergent Functional Genomics (CFG) was usedfor prioritization. Databases of the human gene expression/proteinexpression studies (postmortem brain, peripheral tissue/fluids: CSF,blood and cell cultures), human genetic studies (association, copynumber variations and linkage), and animal model gene expression andgenetic studies, published to date on psychiatric disorders was manuallycurated. Only findings deemed significant in the primary publication, bythe study authors, using their particular experimental design andthresholds, were included in the databases. The databases include onlyprimary literature data and do not include review papers or othersecondary data integration analyses to avoid redundancy and circularity.These large and constantly updated databases have been used in a CFGcross validation and prioritization platform (FIG. 1). For this study,data from 213 papers on AD were present in the databases at the time ofthe CFG analyses (August 2018) (human genetic studies—62, human braintissue studies—49, human peripheral tissue/fluids—83, non-human geneticstudies—4, non-human brain tissue studies—13, non-human peripheraltissue/fluids—2). Analyses were performed as previously described(Niculescu et al., Mol. Psychiatry 2015; 20(11): 1266-1285; Levey etal., Mol. Psychiatry 2016 21(6): 768-785).

Biomarkers to be carried forward were selected after the prioritizationstep, using as threshold a CFG score ≥10 (n=138 probe sets, 112genes).Of these, the top candidate biomarkers had a CFG score ≥12 (n=23 probesets, 18 genes). In Step 3, testing, Low Memory Retention state, andfuture positive neuropsychological testing for cognitive impairment werethen predict in independent cohorts.

In Step 3, testing, the test cohort for predicting Low Memory Retention(state), and the test cohort for predicting Future PositiveNeuropsychological Testing (trait), were assembled out of data that wasRMA normalized by gender and diagnosis. The cohort was completelyindependent from the discovery and validation cohorts, there was nosubject overlap with them. Phenomic (clinical) and gene expressionmarkers used for predictions were Z scored by gender and diagnosis, tobe able to combine different markers into panels and to avoid potentialartefacts due to different ranges of expression in different gender anddiagnoses. Markers were combined by simple summation of the increasedrisk markers minus the decreased risk markers. Predictions wereperformed using R-studio. For cross-sectional analyses, markerexpression levels, z-scored by gender and diagnosis were used. Forlongitudinal analyses, four measures were combined: marker expressionlevels, slope (defined as ratio of levels at current testing visit vs.previous visit, divided by time between visits), maximum levels (at anyof the current or past visits), and maximum slope (between any adjacentcurrent or past visits). For decreased markers, the minimum rather thanthe maximum were used for level calculations. All four measures wereZ-scored, then combined in an additive fashion into a single measure.The longitudinal analysis was carried out in a sub-cohort of the testingcohort consisting of subjects that had at least two test visits.

Predicting State Low Memory. Receiver-operating characteristic (ROC)analyses between marker levels and memory state were performed byassigning subjects visits with a HVLT Retention score of ≤40 into theLow Memory category (using the pROC package of R; Xavier Robin et al.BMC Bioinformatics 2011) (see, FIG. 3). Additionally, a one-tailedt-test was performed between Low Memory group vs. the rest, and PearsonR (one-tail) was calculated between Memory scores and markerlevels.

Predicting Trait Future Positive Neuropsychological Testing forCognitive Impairment. Analyses was conducted for predicting futurepositive neuropsychological testing performed as part of routineclinical care in subjects that had follow-up in the VA system usingelectronic medical records follow-up data of the study subjects (up to12.81 years from initial visit). Analyses between genomic and phenomicmarkers measures (cross-sectional, longitudinal) at a specific testingvisit and future positive neuropsychological test were performed asdescribed below, based on assigning if subjects had a future positiveneuropsychological test for cognitive impairment or not. A Coxregression was performed using the time in days from the lab testingvisit date to the positive neuropsychological testing date. The hazardratio was calculated such that a value greater than 1 always indicatedincreased risk for positive neuropsychological testing, regardless ifthe biomarker was increased or decreased in expression. A hazard ratio(also called odds ratio, O.R.) can be calculated using biomarkerexpression information as a means for predicting risk of futuredevelopment of Alzheimer's and related disorders. Additionally, aPearson R (one-tail) correlation was performed between positiveneuropsychological testing frequency (number of positiveneuropsychological tests divided by duration of follow-up) and markerlevels.

Pharmacogenomics. Which of the top biomarkers from Table 3 (n=38 probesets) known to be modulated by existing drugs were analyzed using theCFG databases, and using Ingenuity Drugs analyses (Tables 2 and 3).

TABLE 2 Top Biomarkers. Convergent Functional Evidence for Relevance toShort-Term Memory Tracking and Alzheimer Disease (AD). Step 1 Step 3Discovery Step 2 Best significant Step 3 in blood External prediction ofBest significant Other Pharmacogenomics (Direction CFG state predictionsof psychiatric Drugs that of change evidence Low memory trait future andrelated modulate tracking for retention positive disorders the increasedinvolve- ROC AUC/ neuropsych evidence biomarker memory) ment in p-valueOR/OR p-value (change in (Change in method/ AD up to 6 pts Up to 6 ptsopposite Same score/ score ALL ALL direction to Direction to CFEGenesymbol/ % up to 4 pts gender 4 pts gender increased Increasedpolyevidence Gene name Probeset up to 6 pts 12 pt 2 pts gender/Dx 2 ptsgender/Dx memory) Memory) score RAB7A  227602_at (I)  7 ALL Gender BPTCA 21 RAB7A, AP/2 L: (17/111) Male Brain Valproate member RAS 43.8% 0.66/1.73E−02 C: (7/91)   arousal oncogene (I) Gender Dx 2.51/3.08E−02depression family DE/4 F-BP MDD 69.6% L: (2/9)    neuropathic   1/2.02E−02 pain M-BP L: (1/27)      1/4.76E−02 M-PSYCHOSIS L:(8/27)    0.76/1.68E−02 M-SZ L: (5/14)     0.8/3.59E−02 M-SZA C:(12/33)   0.67/4.98E−02 NPC2  200701_at (D)  8 ALL Aging 20 Niemann-DE/6 L: (17/111) alcohol Pick disease, 80.8%  0.65/2.38E−02 SZ type C2Gender Male L: (12/79)   0.65/4.65E−02 Gender Dx M-MDD L: (3/18)   0.96/7.58E−03 M-SZA L: (3/13)     0.9/2.13E−02 TGF131  203084_at (I)  9ALL Aging Omega-3 19 transforming AP/4 C: (68/238) ASD fatty acidsgrowth 54.5%  0.58/2.88E−02 BP factor beta 1 Gender Chronic Male stressC: (53/176) Depression   0.6/2.29E−02 Longevity Gender Dx Pain M-PTSDPhencyclidine C: (4/10)   Suicide    1/5.26E−03 PTSD M-SZ SZ C: (15/34)  0.68/3.99E−02 GAP43  204471_at (I)  7 Gender Dx ALL BP Valproate 19growth DE/4 M-SZA C: (11/111) depression Benzodiazepines associated50.8% L: (3/13)   2.07/2.08E−02 SZ protein 43 0.867/3.15E−02 L: (3/50)  stress 6.14/1.51 − 02 Gender Male C: (7/91)   2.94/1.17E−02 L: (3/43)  5.54/1.47 − 02 Gender-Dx M-Psychosis L: (2/22)    5.4/2.96 − 02 M-SZ L:(2/13)   4.08/3.83 − 02 ARSB 1554030_at (I)  6 ALL Alcohol 18arylsulfatase DE/6 L: (17/111) Depression B 91.7%  0.72/2.19E−03 MDDGender Suicide Male L: (12/79)   0.74/4.92E−03 Gender Dx F-BP L:(2/9)     0.93/3.95E−02 M-PSYCHOSIS L: (8/27)    0.88/1.04E−03 M-SZ L:(5/14)     0.8/3.59E−02 M-SZA L: (3/13)       1/5.61E−03 PER1  242832_at(I)  6 Gender Gender Alcohol Lithium 18 period DE/4 Female Male AnxietyClozapine circadian 61.3% C:  (15/62)  L: (3/43)   ASD Quetiapine clock1   0.7/9.17E−03  5.2/4.97E−03 Autism Avibactam Gender Dx BP F-BPCircadian C: (6/19)   abnormalities  0.83/1.13E−02 Depression M-BP MDDL: (1/27)   PTSD     1/4.76E−02 Sleep Duration Suicide SZ GUSB 202605_at (D)  8 ALL Aging Clozapine 18 glucuronidase, DE/4 L: (17/111)Methamphetamine beta 55.7%  0.65/2.16E−02 Gender Female L: (5/32)   0.79/2.29E−02 Gender Dx F-BP C: (6/19)    0.81/1.76E−02 M-MDD L:(3/18)    0.89/1.91E−02 MAPT  203930_s_at (I) 10 ALL Aging Lithium 18microtubule DE/2 L: (11/111) Alcohol Omega-3 associated 33.7%1.96/2.95E−02 Intellect fatty acids protein tau Gender MDD MaleMethamphetamine C: (7/91)   Phencyclidine 3.54/4.62E−02 Stress Gender DxSuicide M-PSYCHOSIS SZ C: (5/47)   2.84/3.34E−02 M-SZ C: (4/27)  4.65/4.06E−02 FCGR1A  216951_at (I)  7 ALL 17 Fc fragment DE/4 L:(3/49)   of IgG, high 64.6%   20/3.50−02 affinity Ia, Gender receptorMale (CD64) L: (3/40)   15.4/4.37E−02 UBE2L3  200682_s_at (D)  4 ALLAging Clozapine 16 ubiquitin DE/6 L: (17/111) Alcohol conjugating 91% 0.63/4.13E−02 ASD enzyme Gender Depression E2L3 Male Stress L: (12/79) SZ  0.65/4.92E−02 Gender Dx M-BP C: (10/54)    0.7/2.25E−02 M-SZA L:(3/13)     0.9/2.13E−02 NKTR 1570342_at (D)  4 ALL Alcohol 16 naturalkiller AP/6 C: (68/238) BP cell 85%  0.59/1.40E−02 Depression triggeringGender MDD receptor Male Social C: (53/176) Isolation  0.62/5.55E−03Stress Gender Dx Suicide M-BP SZ C: (10/54)   0.68/3.56E−02 M-PSYCHOSISC: (27/67)   0.63/3.19E−02 M-PSYCHOSIS L: (8/27)    0.72/3.55E−02 M-SZC: (15/34)   0.72/1.38E−02 M-SZ L: (5/14)       1/1.35E−03 RHEB 243008_at (D)  4 ALL Suicide Antidepressants 16 Ras homolog AP/6 C:(11/111) Pain enriched in 84.4% 1.51/3.05E−02 SZ brain (D) Gender DE/4Male 64.1% C: (7/91)   1.63/2.46E−02 Gender Dx M -PSYCHOSIS C: (5/47)  2.12/5.45E−03 L: (2/22)   9.69/1.68E−02 M-SZ C: (4/27)   1.82/1.78E−02L: (2/13)   6.22/3.32E−02 PTGS2 1554997_a_at (D) 10 Gender Dx AggressionAntipsychotics 16 prostaglandin- DE/4 M-PTSD Alcohol Lithiumendoperoxide 76% C: (4/10) ASD Vorinostat synthase 2  0.88/2.75E−02 BP(prostaglandin Chronic G/H Fatigue synthase and Syndrome cyclooxygenase)Depression Depression- Related MDD Neurological Pain PhencyclidineSocial Isolation Stress Stress Substances/ Addictions Suicide RGS10 214000_s_at (I)  6 ALL Aging 16 regulator of DE/4 L: (17/111) BPG-protein 63.5%   0.7/3.89E−03 Female signaling 10 Gender specific Maleinterpersonal- L: (12/79)  traumas  0.74/4.73E−03 Methamphetamine GenderDx Post- F-BP Deployment L: (2/9)    PTSD  0.93/3.95E−02 PTSD M-BPStress L: (1/27)   Suicide     1/4.76E−02 SZ M-MDD L: (3/18)   0.87/2.53E−02 M-SZ C: (15/34)   0.68/3.70E−02 MAPT  203928_x_at (I) 10Gender Dx Aging Lithium 16 microtubule DE/4 F-BP Alcohol Omega-3associated 57.5% C: (6/19)   Intellect fatty acids protein tau 0.81/1.76E−02 MDD Methamphetamine Phencyclidine Stress Suicide SZ ITPKB 232526_at (I)  6 ALL Aging 16 inositol- DE/4 L: (17/111) Alcoholtrisphosphate 51.9%  0.73/1.60E−03 MDD 3-kinase B Gender PhencyclidineMale Stress L: (12/79)  Suicide, SZ   0.7/1.44E−02 SZ Female L: (5/32)   0.79/2.29E−02 Gender Dx M-BP L: (1/27)       1/4.76E−02 KIDINS220 214932_at (I)  6 Gender Dx Gender Alcohol Clozapine 16 kinase D- DE/4F-BP Male MDD interacting 51.9% L: (2/9)    C: (7/91)   Psychosissubstrate  0.93/3.95E−02 2.49/3.78E−02 Pain 220 kDa Gender-Dx SuicideM-BP Stress C: (2/16)   6.06/4.18 − 02 GSK3B  209945_s_at (D) 10 GenderDx Aging Astaxanthin- 16 glycogen DE/4 M-SZA Alcohol DHA synthase 50.3%L: (3/13)   ASD Antipsychotics kinase 3 beta  0.93/1.40E−02 BP LithiumBP, SZ Omega-3 MDD fatty acids Stress Ketamine Suicide lipoteichoic SZacid Valproate enzastaurin, glycogen synthase kinase-3beta inhibitorSERTAD3  219382_at (D)  5 Gender Alcohol 15 SERTA DE/6 Female ASD domain81.4% L: (5/32)   Aging containing 3  0.79/2.29E−02 Gender Dx F-BP C:(6/19)    0.81/1.76E−02 F-PSYCHOSIS L: (2/13)       1/1.50E−02 F-SZA L:(2/8)        1/2.28E−02 APOE  212884_x_at (D) 11 Gender Dx AggressionOmega-3 15 apolipoprotein AP/2 M-PTSD Aging fatty acids E 34.1% C:(4/10)   Alcohol  0.88/2.75E−02 Anxiet Gender Dx ASD M-SZ BP L: (5/14)  Brain  0.89/9.82E−03 arousal MDD PTSD Stress Suicide SZ TBI UBE2I 233360_at (D)  6 Gender Dx Aging Clozapine 14 ubiquitin DE/6F-PSYCHOSIS Alcohol conjugating 86.8% L: (2/13)   ASD enzyme E21 0.91/3.78E−02 Hallucinations F-SZA Mood State L: (2/8)    Stress 0.92/4.78E−02 FOXO3  231548_at (I)  4 Gender Dx Gender Dx BP Clozapine14 forkhead box AP/2 F-SZA M-PSYCHOSIS Cocaine O3 38.9% C: (5/15)   C:(5/47)   Longevity (I)  0.78/4.32E−02 4.14/4.58E−02 PTSD DE/6 Stress82.3% Suicide THRA  214883_at (I)  8 Gender Dx Alcohol 3,5- 14 thyroidDE/4 F-BP PTSD diiodothyropropionic hormone 61.3% C: (6/19)   Stressacid,denosum receptor,  0.79/2.18E−02 Suicide ab/levothyrox alpha M-BPSZ ine,amiodaro L: (1/27)   ne,levothyrox     1/4.76E−02 ine,dextrothyroxine,L- triiodothyronine ITPKB 1554306_at (D)  6 Gender Acute Omega-314 inositol- AP/4 Female Stress fatty acids trisphosphate 61.1% L:(5/32)   Aging 3-kinase B (D)  0.81/1.37E−02 Alcohol DE/4 Gender Dx ASD55.7% F-BP BP C: (6/19)   MDD  0.91/2.50E−03 Neurological F-BP SuicideL: (2/9)    SZ     1/2.02E−02 IGF1  209542_x_at (I)  8 Gender DxAggression Lithium 14 insulin-like DE/4 F-BP Aging Clozapine growth54.1% C: (6/19)   Alcoho Fluoxetine factor 1  0.79/2.18E−02 Anxiety(SSRI), (somatomedin BP Venlafaxine C) Depression (SNRI) LongevityMEDI-573, PTSD BI836845 SZ NPTX2  213479_at (I)  8 Gender Dx AlcoholClozapine 14 neuronal DE/4 F-BP Brain Fluoxetine pentraxin II 52.5% L:(2/9)    arousal  0.93/3.95E−02 Cocaine Depression MDD MDD, SZ MoodDisorders NOS Stress Suicide GSTM3  235867_at (D)  8 Gender Dx BP 14glutathione DE/4 F-SZA MDD S- 52.1% C: (5/15)   SZ transferase 0.78/4.32E−02 mu 3 (brain) BACE1  222463_s_at (I)  8 Gender MDD 14Beta- DE/2 Male Stress Secretase 1 44.8% C: (7/91)   Suicide1.97/3.78E−02 PSEN1  203460_s_at (D)  9 Aging Omega-3 13 presenilin 1DE/4 Alcohol fatty acids 54.5% Autism Depression Emotional StabilityNeuroticism Suicide SZ GFAP  203540_at (I)  9 Gender Dx AddictionsOmega-3 13 glial DE/2 F-BP Alcohol fatty acids fibrillary 34.3% C:(6/19)   BP Clozapine acidic protein  0.77/3.28E−02 MDD Stress SuicideSZ Yohimbine TREM2  219725_at (I) 11 BP 13 triggering DE/2 SZ receptor37.6% expressed on myeloid cells 2 NOCT  220671_at (D)  6 Gender Dx PTSD12 nocturnin AP/4 F-PTSD Post- 69.5% C: (3/9)    Deployment    1/1.01E−02 PTSD CEP350  204373_s_at (D)  6 Gender Dx AutismAntidepressants, 12 centrosomal DE/4 M-PSYCHOSIS BP Fluoxetine protein67.1% L: (2/22)   Cocaine 350 kDa 54.6/3.77E−02 Depression PTSD StressSuicide SZ PPP2R2B  205643_s_at (I)  6 Gender Dx ADHD 12 protein DE/4F-BP Aging phosphatase 63.5% L: (2/9)    Alcohol 2, regulatory    1/2.02E−02 ASD subunit B, Circadian beta abnormalities LongevityPTSD Suicide SZ NRP2  222877_at (I)  6 Gender Dx Longevity Clozapine 12neuropilin 2 DE/4 M-MDD MDD 61.3% L: (3/18)   Phencyclidine 0.98/5.43E−03 Stress CTSS  232617_at (D)  8 Aging Omega-3 12 cathepsinS DE/4 Alcohol fatty acids 56.9% ASD BP Brain arousal Pain Suicide VEGFA 211527_x_at (I)  8 Gender Dx Alcohol Antipsychotics 12 vascular DE/2M-MDD Anxiety Fluoxetine endothelial 45.3% C: (11/38)  BP Steroidsgrowth factor   0.7/2.57E−02 Chronic A Stress Depression HallucinationsIntellect MDD . Pain MSK Stress Suicide SZ MAPT  233117_at (I) 10 AgingLithium 12 microtubule DE/2 Alcohol Omega-3 associated 44.2% Intellectfatty acids protein tau MDD Methamphetamine Phencyclidine Stress SuicideSZ GSK3B  240562_at (I) 10 Aging Antipsychotics 12 glycogen DE/2 AlcoholAntipsychotics synthase 39.2% ASD Pregnenolone kinase 3 beta BP sulfateMDD Fluoxetine Methamphetamine (SSRI) Psychological Stress LithiumStress mood Suicide stabilizing SZ drugs Yohimbine Valproate GS1C3B 242336_at (D) 10 Aging Astaxanthin- 12 glycogen AP/2 Alcohol DHAsynthase 34.1% ASD Antipsychotics kinase 3 beta BP Lithium BP,SZ Omega-3MDD fatty acids Stress Ketamine Suicide lipoteichoic SZ acid Valproateenzastaurin, glycogen synthase kinase-3beta inhibitor BACE1  224335_s_at(I)  8 MDD 10 Beta- DE/2 Stress Secretase 1 43.1% Suicide Bold-topbiomarkers after discovery and prioritization (n = 23, CFG ≥ 12)).Underlined-best predictor in a category after testing of the longer listcandidate biomarkers after discovery and prioritization (n = 138, CFG ≥10), as depicted in Figure 3. We tabulated into a convergent functionalevidence (CFE) score all the evidence from discovery (up to 6 points),prioritization (up to 12 points), testing (State Memory Retention Stateand Trait Future Positive Neuropsychological Testing (up to 6 pointseach if significantly predicts in all subjects, 4 points if predicts bygender, 2 points if predicts in gender/diagnosis subgroups). The goal isto highlight, based on the totality of our data and of the evidence inthe field to date, biomarkers that have all around evidence: trackmemory, are implicated in AD, and predict memory state and futuredementia. Such biomarkers merit priority evaluation in future clinicaltrials. As depicted in Figure 1B, the top row of values-increased inexpression (I) in high memory, bottom row of values-decreased inexpression (D) in high memory. DE-differential expression,AP-Absent/Present. “C”-Cross-sectional analyses; “L”-Longitudinalanalyses, using levels and slopes from multiple visits. In All, byGender, and personalized by Gender and Diagnosis (Gender/Dx).“DE”-differential expression, “AP”-Absent/Present. For Step 3Predictions, C-cross-sectional (using levels from one visit),L-longitudinal. “M”-males, “F”-Females. “MDD”-depression, “BP”-bipolar,“SZ”-schizophrenia, “SZA”-schizoaffective, PSYCHOSIS-schizophrenia andschizoaffective combined, “PTSD”-post-traumatic stress disorder.

TABLE 3 Matching with drugs. Evidence for modulation by drugs in samedirection as increased memory retention (see also, FIG. 5). Step 1Discovery in Blood Step 2 (Direction External of Change CFG trackingEvidence Memory For Increase Involvement Method/ in AD Genesymbol/Score/% Score Anti- Gene name Probesets Up to 6 pts Up to 12 pts LithiumOmega-3 depressants Other drugs APOE  212884_x_at (D) 11 (D)apolipoprotein AP/2 Lymphocytes E 34.1% (males) Omega-3 fatty acids³¹⁸GSK3B  242336_at (D) 10 (D) (D) (D) glycogen  209945_s_at AP/2 olfactoryPFC HIP synthase 34.1% neurons (females) Ketamine³²⁰ kinase (D)Lithium²⁹⁸ Omega-3 (D) 3 beta DE/4 Lithium³¹⁹ fatty acids³¹⁸ HIP 50.3%(D) lipoteichoic acid³⁰¹ HIP (D) Alzheimer's Disease Caudate putamenAstaxanthin-DHA⁴³ Valproate²²² (D) Frontal Cortex Antipsychotics³²¹Enzastaurin MAPT  203930_s_at (I) 10 (I) (I) microtubule  233117_at DE/2Schneider HIP associated  203928_at 33.7% 2 (S2) cells (males) proteintau (I) Lithium³²² Omega-3 DE/2 fatty acids³¹⁸ 44.2% (I) DE/4 57.5%PTGS2 1554997_a_at (D) 10 (D) (D) prostaglan DE/4 PBMC Serum, HIPdinendoperoxide 76% Lithium¹⁶⁵ Vorinostat³²³ synthase 2 (D)(prostaglandin PBMC G/H synthase Antipsychotics¹⁶⁵ and Acetaminophencyclooxygenase) NSAIds GFAP  203540_at (I)  9 (I) (I) glial fibrillaryDE/2 Brain AMY, HIP, PFC acidic protein 34.3% Omega-3 Clozapine¹⁷¹ fattyacids³²⁴ PSEN1  203460_s_at (D)  9 (D) tarenflurbil presenilin 1 DE/4Lymphocytes 54.5% (females) Omega-3 fatty acids³¹⁸ TGFB1  203084_at (D) 9 (D) dalantercept, transforming AP/4 Lymphocytes fresolimumab, growthfactor 54.5% (females) LY3200882, beta 1 Omega-3 MSB0011359C fattyacids³¹⁸ BACE1  222463_s_at (I)  8 Beta-Secretase 1  224335_s_at DE/644.8% (I) DE/6 43.1% CTSS  232617_at (D)  8 (D) cathepsin S DE/4Lymphocytes 56.9% (females) Omega-3 fatty acids³¹⁸ GUSB  202605_at (D) 8 (D) glucuronidase, DE/4 VT beta 55.7% Clozapine¹⁷¹ IGF1  209542_x_at(I)  8 (I) (I) (I) insulin-like DE/4 lymphoblastoid HIP VT growth factor1 54.1% cell lines Fluoxetine Clozapine¹⁷¹ (somatomedin C) Lithium³²⁵(SSRI), Venlafaxine (SNRI)³²⁶ NPTX2  213479_at (I)  8 (I) (I) neuronalDE/4 HIP VT pentraxin II 52.5% Fluoxetine³²⁷ Clozapine¹⁷¹ THRA 214883_at (I)  8 thyroxine thyroid hormone DE/4 receptor, alpha 61.3%VEGFA  211527_x_at (I)  8 (I) (I) vascular DE/2 Cortex Plasmaendothelial 45.3% Fluoxetine³²⁸ Antipsychotics²⁰⁴ growth factor A (I)Blood Steroid³²⁹ GAP43  204471_at (I)  7 (I) growth associated DE/4Human protein 43 50.8% astrocyte-derived cells (U-87 MG) Valproate³³⁰(I) HIP Benzodiazepines³³¹ RAB7A  227602_at (I)  7 (I) (I) RAB7A, memberAP/2 basal forebrain Caudate putamen RAS oncogene 43.8% TCA³³²Valproate²²² family (I) DE/4 69.6% KIDINS220  214932_at (I)  6 (I)kinase DE/4 VT D-interacting 51.9% Clozapine¹⁷¹ substrate 220 kDa CD36 242197_x_at (D)  6 (D) CD36 molecule DE/4 Lymphocytes (thrombospondin67.1% Benzodiazepines³³¹ receptor) CEP350  204373_s_at (D)  6 (D)centrosomal DE/4 AMY protein 67.1% Antidepressants, 350 kDaFluoxetine²²⁵ ITPKB 1554306_at (D)  6 (D) inositol- AP/4 lymphocytestriphosphate 61.1% (males) 3-kinase B (D) Omega-3 DE/4 fatty acids³¹⁸55.7% NRP2  222877_at (I)  6 (I) neuropilin 2 DE/4 CP 61.3% Clozapine¹⁷¹PER1  242832_at (I)  6 (I) (I) period circadian DE/4 Cerebral VT clock 161.3% cortex (right) Clozapine¹⁷¹ Lithium³³³ (I) (I) AMY lymphoblastoidQuetiapine³³⁵ cell lines (LCLs) derived Lithium³³⁴ UBE21  233360_at (D) 6 (D) ubequitin DE/6 VT conjugating 86.8% Clozapine¹⁷¹ enzyme E2I FOXO3 231548_at (I)  4 (I) forkhead AP/2 Lymphocytes, box O3 38.9% VTClozapin¹⁷¹ (I) DE/6 82.3% RHEB  243008_at (D)  4 (D) Ras homolog AP/6NR1³³⁶ enriched in brain 84.4% (D) DE/4 64.1% UBE2L3  200682_s_at (D)  4(D) ubiquitin DE/6 VT conjugating 91% Clozapine¹⁷¹ enzyme E2L 3

Tables 4A & 4B. Methods for Personalized Assessment of Memory State(Table 4A) and Prediction of Future Risk for Alzheimer and RelatedDisorders (Table 4B). Personalized by Gender and Psychiatric Diagnosis.

M—males; F—females; BP—bipolar; MDD—Major Depressive Disorder;PTSD—Post-Traumatic Stress Disorder; PSYCHOSIS—schizophrenia orschizoaffective disorder; SZ—schizophrenia; SZA—schizoaffectivedisorder; I—increased; D—decreased.

N—

TABLE 4A Assessment for Memory State Direction of Change in LowDiagnosis Best Individual Biomarker Memory M-BP NAV2 D M-BP UBE2L3 IM-MDD CD40 I M-MDD LOC101928123 D M-PSYCHOSIS ARSB D M-PTSD TGFB1 I M-SZNKTR I M-SZA ARSB D M-SZA CD36 I F-BP CACNA1S D F-BP ITPKB I F-PSYCHOSISSERTAD3 I F-PSYCHOSIS LINC01398 D F-PTSD NOCT I F-SZA SERTAD3 I F-SZALINC01398 D

TABLE 4B Prediction of Future Risk for Alzheimer's and Related DisordersDirection of Change in Low Diagnosis Best Individual Biomarker MemoryM-BP KIDINS220 D M-PSYCHOSIS CEP350 I M-PSYCHOSIS CALHM1 D M-SZ RHEB IM-SZ MAPT DTables 5A-5C. New Therapeutics. Discovery of new method of use fordrugs/repurposing. Table 5A. Connectivity Map (CMAP) analysis. Query forsignature is done using exact Affymetrix probe sets and direction ofchange. Drugs that have same gene expression profile effects to our highmemory retention biomarkers signatures. A score of 1 indicates theperfect match, i.e. the best potential therapeutic for increasing memoryretention. Table 5B. NIH LINCS analysis using the L1000CDS2 (LINCS L1000Characteristic Direction Signature Search Engine) tool. Query forsignature is done using gene symbols and direction of change. Shown arecompounds mimicking direction of change in high memory. A higher scoreindicates a better match. Table 5C. CRowd Extracted Expression ofDifferential Signatures (CREEDS) analysis. Query for signature is doneusing gene symbols and direction of change. Shown are compoundsmimicking direction of change in high memory. A higher score indicates abetter match.Table 5A. Drug repurposing using Connectivity Map (CMAP from BroadInstitute/MIT)

TABLE 5A1 Drugs Identified Using Gene Expression Panels of TopBiomarkers CFG ≥ 12 (n = 23 probe sets; 7 increased and 6 decreased werepresent in HG-U133A array used by CMAP). Panel of genes increased inexpression: MAPT (2 probe sets), TREM2, GFAP, THRA, IGF1, NPTX2 Panel ofgenes decreased in expression: NPC2, GSK3B, GUSB, TGFB1, APOE, PSEN1rank CMAP name score Description 1 verteporfin 1 A benzoporphyrinderivative, it is a medication used as a photosensitizer forphotodynamic therapy to eliminate the abnormal blood vessels in the eyeassociated with conditions such as the wet form of macular degeneration.2 pioglitazone 0.987 A drug of the thiazolidinedione (TZD) class withhypoglycemic (antihyperglycemic, antidiabetic) action, used to treatdiabetes. PPAR gamma agonist. There is evidence to suggest piolitazoneis associated with a lower risk of dementia in type 2 diabetics. Phase 3clinical trials failed to meet endpoints using pioglitazone as atherapeutic for MCI/AD. 3 salsolidine 0.972 A tetrahydroisoquinolineisolated from plants of the genus Salsola. Tetrahydroisoquinolines aresteroselective competitive inhibitors of the enzyme MAO. They are also acompetitive inhibitors of COMT. 4 sulfadimidine 0.97 A sulfonamideantibacterial. 5 SB-203580 0.968 Specific inhibitor of p38MAPK. 6ronidazole 0.966 An antiprotozoal agent used in veterinary medicine. 7mesalazine 0.961 Anti-inflammatory salycilate derivative used to treatulcerative colitis. 8 dioxybenzone 0.946 An organic compound used insunscreen to block UVB and short-wave UVA rays. It is a derivative ofbenzophenone. 9 metamizole 0.942 A non-steroidal anti-inflammatory drug.10 8-azaguanine 0.936 A purine analog with antineoplastic activity. 11sulfaphenazole 0.935 A long-acting sulfonamide antibiotic used in thetreatment of leprosy. 12 dicoumarol 0.933 A naturally occurringanticoagulant drug that depletes stores of vitamin K. In general,vitamin K antagonists may have a negative influence on visual memory,verbal fluency, and brain volume. 13 tolazamide 0.915 Anintermediate-acting, first-generation sulfonylurea with hypoglycemicactivity. 14 pipemidic acid 0.911 A member of the pyridopyrimidine classof antibacterials. 15 NS-398 0.911 A COX-2 inhibitor. May acutelyprevent the suppression of hippocampal long-term plasticity by amyloidbeta. 16 morantel 0.901 An anthelmintic drug used for the removal ofparasitic worms in livestock. An inhibitor of acetylcholinesterase. 17indapamide 0.901 A thiazide-like diuretic drug generally used in thetreatment of hypertension, as well as decompensated heart failure.Indapamide has been shown to suppress the production of amyloid beta andimprove clearance. 18 promazine 0.893 Blocks postsynaptic dopaminereceptors D1 and D2 in the mesolimbic and medullary chemoreceptortrigger zone. Has significant interaction with multiple Alzheimer targetproteins. 19 tinidazole 0.893 A nitroimidazole antitrichomonal agenteffective against Trichomonas vaginalis, Entamoeba histolytica, andGiardia lamblia infections. 20 estradiol 0.892 An estrogen steroidhormone. There is evidence that suggests lifetime exposure to estrogenseems to lower risk of AD. Women who began estradiol treatment withinone year of menopause had preserved metabolic activity in regions in andaround the hippocampus. It is unclear whether above the age of 50 years,if estrogen/estradiol is protective against AD.

TABLE 5A2 Drugs Identified Using Gene Expression Panels of TopBiomarkers CFG ≥ 10 (n = 138 probe sets; 45 increased and 38 decreasedwere present in HG-U133A array used by CMAP). Panel of genes increasedin expression: BCAM, HFE, SLC1A7, FTL, MAPT, GFAP, LDLR, SNCA, THRA,C4A, TREM2, CSF1, SNCA, VEGFA, IL1A, SNCA, CSF1, NRP2, GAP43, CHAT,KIDINS220, NPTX2, ANK1, IGF1, IGHG1, MAPT, FXYD1, LMNA, ANK1, IGHG1,AXL, THRA, PPP2R2B, ANK1, RGS10, FCGR1A, LMNA, ITGB5, APOA1, ZBTB16,OPHN1, ARG2, TSPAN5, AIMP2, RPL38. Panel of genes decreased inexpression: APOE, VEGFA, HSPA5, ZFP36L1, TGFB1, NDUFA5, DKK1, NOCT,WDR45, IGF1, CSF1R, ICAM1, VEGFA, ABCA7, GSK3B, GAPDH (2), SREBF1,DUSP6, UQCRC1, TPK1, MICA, PSEN1, PSMA4, GUSB, NDUFS3, BST2, TYROBP,CEP350, FDPS, MTF2, NPC2, SERTAD3, HSBP1, SEC24A, SNRK, TRIM38, UBE2L3.rank CMAP name score Description 1 levonorgestrel 1 Progesteronederivative used as contraceptive. Progesterone and its derivatives havesome evidence for promoting brain cell growth, at least in adult rats,and some studies have shown that it can improve cognitive performance inthe aging mouse. 2 aminohippuric 0.955 Non-toxic diagnostic tool tomeasure effective renal plasma acid flow. 3 meglumine 0.933 Meglumine,also known as megluminum or methylglucamine, belongs to the class oforganic compounds known as hexoses. Often used as an excipient inpharmaceuticals. Methylglucamine orotate is a memory- improving drug,although the ortoate component was thought to be the active compound. 4mesalazine 0.932 Non-steroidal anti-inflammatory drug used to treatinflammatory bowel diseases. 5 lymecycline 0.92 Tetracycline antibiotic;tetracyclines have been shown to have beneficial effects inneurodegenerative diseases. 6 torasemide 0.918 Diuretic. 7 dioxybenzone0.916 Sunscreen compound. 8 ginkgolide A 0.915 A natural compound withneuroprotective and possible AD preventing effects. 9 rimexolone 0.907Rimexolone is a derivative of prednisolone, a synthetic glucocorticoidwith anti-inflammatory and immunosuppressive property. 10 ketanserin0.905 Ketanserin is a selective serotonin receptor antagonist with weakadrenergic receptor blocking properties. Effective in lowering bloodpressure in essential hypertension. Also inhibits platelet aggregation.Well tolerated in older patients. 11 dicloxacillin 0.903 APenicillin-class antibacterial. 12 talampicillin 0.898 A beta lactamantibiotic from the penicillin family. 13 sulfadimidine 0.897 Asulfonamide antibacterial. 14 naringin 0.892 Naturally occurringflavinoid in citrus fruits, especially grapefruit. There is evidence instudies with rats that narigin acts through inhibition of oxidativecellular stress which attenuates autophagic stress especially in thehippocampus. Furthermore, there is evidence that ICV-STZ ratschronically treated with naringin dose dependently restored cognitivedeficits. 15 naproxen 0.891 Nonsteroidal anti-inflammatory drug. Severallarge scale studies have demonstrated that long term treatment withnaproxen confers no protection against cognitive decline. 16 flunixin0.888 A nonsteroidal anti-inflammatory drug, analgesic, and antipyreticused in horses, cattle and pigs. 17 tubocurarine 0.887 A neuromuscularblocker and active ingredient in curare; chloride plant based alkaloidof Menispermaceae. There is evidence that anticholinergics in generalare associated with future incidence of dementia. 18 cyanocobalamin0.885 Vitamin B12. There is evidence that increased plasma levels ofhomocysteine (which can be caused by low levels of vitamin B12) is astrong and independent risk factor for the development of dementia andAD. 19 dequalinium 0.883 A topical bacteriostat. There is evidence thatdequalinium chloride induces protofibril formation of alpha-synuclein.20 meticrane 0.882 A sulphonamide-derivative with thiazide-like diureticactivity.

TABLE 5A3 Drugs Identified Using Gene Expression Panels of PredictiveBiomarkers in All (n = 16 probe sets/genes; 5 increased and 11 decreasedwere present in HG-U133A array used by CMAP). Panel of genes increasedin expression: FCGR1A, GAP43, MAPT, HFE, RGS10, Panel of genes decreasedin expression: NDUFA5, SEC24A, PSMA4, UBE2L3, NPC2, GUSB, TGFB1, TRIM38,CD40, ZNF345, IGF1. rank CMAP name score Description 1 mesalazine 1Non-steroidal anti-inflammatory drug used to treat inflammatory boweldiseases. 2 mepenzolate 0.985 An oral, quaternary anticholinergicgastrointestinal agent bromide used for adjunctive treatment of pepticulcer disease. 3 ozagrel 0.974 Antiplatelet agent working as athromboxane A2 synthesis inhibitor. Commonly used in the treatment ofstroke. 4 protriptyline 0.954 A tricyclic antidepressant that increasesthe synaptic concentration of serotonin and/or norepinephrine. In vitro,protriptyline has been shown to inhibit acetylcholinesterase,β-secretase, amyloid β aggregation, and glycation induced amyloidaggregation-all causal factors in AD progression (Bansode et al. 2014) 5guanfacine 0.945 A selective alpha_(2A)-adrenoreceptor agonist that isused as an antihypertensive. It also preferentially binds postsynapticalpha_(2A)-adrenoreceptors in the prefrontal cortex which allows its usein improving symptoms associated with ADHD. It is not a CNS stimulant. 6saquinavir 0.94 An anti-retroviral protease inhibitor commonly used inthe treatment of HIV. 7 tomatidine 0.938 A steroidal alkaloid that hasbeen found in the skins and leaves of tomatoes. It suppresses NF-κBsignaling in LPS- stimulated macrophages, blocking induced expression ofinducible nitric oxide synthase and COX-2. 8 eldeline 0.936 9zuclopenthixol 0.931 An antipsychotic agent working as an antagonist atD1 and D2 dopamine receptors. 10 fenoterol 0.929 A synthetic adrenergicβ₂-agonist that is used as a bronchodilator and tocolytic. 11 vincamine0.929 A monoterpenoid indole alkaloid obtained from the leaves of Vincaminor with a vasodilatory property. 12 imipenem 0.926 A carbapenemantibacterial. 13 isradipine 0.924 A second generation calcium channelblocker that is used to treat hypertension. 14 3-hydroxy-DL- 0.919 Ametabolite of tryptophan, which filters UV light in the kynurenine humanlens. 15 amiodarone 0.912 A class III antiarrhythmic agent, amiodaroneblocks the myocardial calcium, potassium and sodium channels in cardiactissue, resulting in prolongation of the cardiac action potential andrefractory period. In addition, this agent inhibits alpha- andbeta-adrenergic receptors, resulting in a reduction in sympatheticstimulation of the heart, a negative chronotropic effect, and a decreasein myocardial oxygen demands. 16 lansoprazole 0.911 A proton pumpinhibitor (PPI) and a potent inhibitor of gastric acidity. 17 nialamide0.911 A non-selective, irreversible monoamine oxidase inhibitor of thehydrazine class that was used as an antidepressant. It was withdrawn byPfizer several decades ago due to the risk of hepatotoxicity. 18hydralazine 0.909 An antihypertensive with vasodilatory effects. 19S-propranolol 0.906 The active enantiomer of propranolol, a β-adrenergicreceptor antagonist. 20 nomifensine 0.906 A norepinephrine-dopaminereuptake inhibitor.

TABLE 5A4 Drugs Identified Using Gene Expression Panels of PredictiveBiomarkers in Males (n = 17 probe sets/genes; 6 increased and 11decreased were present in HG-U133A array used by CMAP). Panel of genesincreased in expression: FCGR1A, GAP43, MAPT, KIDINS220, AIMP2, RGS10Panel of genes decreased in expression: NDUFA5, SEC24A, PSMA4, UBE2L3,NPC2, BST2, TGFB1, TRIM38, ZNF345, IGF1, VEGFA rank CMAP name scoreDescription 1 natamycin 1 Ophthalmic antifungal suspension. 2mepenzolate 0.9 An oral, quaternary anticholinergic gastrointestinalagent bromide used for adjunctive treatment of peptic ulcer disease. 3valinomycin 0.896 A natural antibiotic derived from Streptomyces. Italso binds potassium ions and facilitates their transfer across lipidbilayers. 4 aminohippuric 0.881 Non-toxic diagnostic tool to measureeffective renal acid plasma flow. 5 dexpropranolol 0.859 A non-selectiveβ-adrenergic blocker. Studies have shown propranolol reduces cognitivedeficits and amyloid/tau pathology in AD simulated mice. 6 valproic acid0.851 A histone deacetylase inhibitor commonly used as an anticonvulsantand antimanic agent. Studies show valproic acid enhances memory andcognition in mice models. 7 dicloxacillin 0.85 A penicillin antibiotic.8 pronetalol 0.837 An early non-selective β-blocker candidate that wasnot used clinically as it formed a carcinogenic metabolite in mice. 9iobenguane 0.837 A guanidine analog with specific affinity for tissuesof the sympathetic nervous system. The radiolabeled forms are used asantineoplastic or radioactive imaging agents. May be useful fordiagnosing AD or dementia with Lewey bodies. 10 todralazine 0.829 Anantihypertensive agent with central and peripheral action. It has someCNS depressant effects as well. 11 torasemide 0.827 An anilinopyridinesulfonylurea belonging to the class of loop diuretics. 12 gallamine0.824 A non-depolarising muscle relaxant. It acts by combiningtriethiodide with the cholinergic receptor sites in muscle andcompetitively blocking the transmitter action of acetylcholine. 13sulconazole 0.822 An antifungal medication of the imidazole class. 14chlormezanone 0.82 A non-benzodiazepine muscle relaxant. It wasdiscontinued worldwide in 1996 due to rare but serious cases of toxicepidermal necrolysis. 15 amantadine 0.818 A primary amine that has bothantiviral and dopaminergic activity and is used in the therapy ofinfluenza A and management of Parkinson disease. 16 tubocurarine 0.818 Aneuromuscular blocker and active ingredient in curare; chloride plantbased alkaloid of Menispermaceae. 17 protriptyline 0.805 A tricyclicantidepressant. 18 indometacin 0.8 A nonsteroidal anti-inflammatory drug(NSAID). 19 thioguanosine 0.799 A thio analogue of the naturallyoccurring purine base guanine used to treat acute myeloid leukemia,acute lymphocytic leukemia, and chronic myeloid leukemia. 20 adenosine0.797 A nucleotide that is found in RNA. phosphate

TABLE 5A5 Drugs Identified Using Gene Expression Panels of PredictiveBiomarkers in Females (n = 13 probe sets/genes; 1 increased and 4decreased were present in HG-U133A array used by CMAP). Panel of genesincreased in expression: CHAT Panel of genes decreased in expression:GUSB, CD40, SERTAD3, TBRG4 rank CMAP name score Description 1benserazide 1 Peripherally acting aromatic L-amino acid decarboxylase orDOPA decarboxylase inhibitor, which is unable to cross the blood-brainbarrier. Recent studies by Jonkers et al. and Shen et al. revealed thatbenserazide can enter the brain and affect levodopa metabolism. 2 TTNPB0.99 Selective and highly potent retinoic acid analog with affinity forretinoic acid receptors (RAR) α, β, and γ, which are nucleartranscription factors. Activation of RAR and RXR is known to impede thepathogenesis of AD in mice by inhibiting accumulation of amyloids. 3suxibuzone 0.979 Analgesic used for joint and muscular pain. 4 15-delta0.962 Prostaglandin J derivative. It has a role as a metabolite,prostaglandin J2 an electrophilic reagent and an insulin-sensitizingdrug. Koma et al. found 15d-PGJ2-impaired memory retrievalsignificantly. Pereira et al. concluded therapeutic potential oftargeting the J2 prostaglandin pathway to prevent/delayneurodegeneration associated with neuroinflammation 5 hydroquinine 0.961Anti-arrhythmia agent and parasympatholytic. 6 rosiglitazone 0.954 Anantidiabetic drug in the thiazolidinedione class. It works as an insulinsensitizer, by binding to the PPAR in fat cells and making the cellsmore responsive to insulin. Rosiglitazone reverses memory decline andhippocampal glucocorticoid receptor down-regulation in an Alzheimer'sdisease mouse model (Escribano 2009). In Phase 2 clinical trials fordetermining role in learning and memory in patients diagnosed with MCI.7 colchicine 0.942 An anti-inflammatory which acts by inhibition ofmicrotubule polymerization. Impairs memory function in a dose-dependentmanner and is used as a model to induce Alzheimer's disease in rats. 82,6- 0.942 — dimethylpiperidine 9 primaquine 0.939 An antimalarial agentthat acts by interfering with the mitochondria of parasites. 10 15-delta0.931 Prostaglandin J derivative. It has a role as a metabolite,prostaglandin J2 an electrophilic reagent and an insulin-sensitizingdrug. Koma et al. found 15d-PGJ2-impaired memory retrievalsignificantly. Pereira et al. concluded therapeutic potential oftargeting the J2 prostaglandin pathway to prevent/delayneurodegeneration associated with neuroinflammation 11 meropenem 0.925Carbapenem antibiotic. 12 anabasine 0.924 A nicotine analog that is analkaloid. Has demonstrated improvement in memory and attention in rats.13 cyclizine 0.919 A piperazine-derivative antihistamine used as anantivertigo/antiemetic agent. 14 norcyclobenzaprine 0.919 A metaboliteof cyclobenzaprine (a muscle relaxant). 15 naftopidil 0.918 Anα1-adrenergic receptor antagonist. 16 BAS-012416453 0.914 — 17 AG-0125590.912 — 18 terbutaline 0.91 A β2 adrenergic receptor agonist. 19clomipramine 0.908 A tricyclic antidepressant used in the therapy ofobsessive-compulsive disorder. Associated with diminished metamemory andimpaired priming and working memory. 20 methyldopa 0.904 Anantihypertensive that is a competitive inhibitor of the enzyme DOPAdecarboxylase which converts L-DOPA into dopamine. Has been associatedwith verbal memory impairment.Table 5B. Drug repurposing using L1000 Characteristic DirectionSignature Search Engine.

TABLE 5B1 Drugs Identified Using Gene Expression Panels of TopBiomarkers CFG ≥ 12 (n = 18 unique genes; 8 increased and 10 decreased).Panel of genes increased in expression: MAPT, GFAP, TREM2, ARSB, IGF1,THRA, NPTX2 BACE1 Panel of genes decreased in expression: GSK3B, NPC2,PTGS2, PSEN1, CTSS, GSTM3, UBE2I, GUSB, APOE, TGFB1 Rank Score DrugDescription 1 0.2778 BRD-K03371390 2 0.2778 NCGC00185923-01 3 0.2222BENZANTHRONE Dye that binds to amyloid fibrils. 4 0.2222 SQ 22536Adenylyl cyclase inhibitor. 5 0.2222 ICARIIN Prenylated flavanolglycoside from Epimedium sagittatum. Jin et al. 2014 has found thatIcariin significantly improved learning and memory of transgenic micemodels of AD via stimulation of the NO/cGMP pathway. Sheng et al. 2017concluded that Icariin improves synaptic plasticity, and thereforelearning and memory, in rat models of AD via the BDNF/TrkB/Akt pathway.6 0.2222 YM 90709 IL-5 receptor antagonist. 7 0.2222 QUIPAZINE Binds toserotonin receptors, particularly to MALEATE 5HT2A and 5HT3. 8 0.2222Cisapride Serotonin 5-HT₄ receptor agonist. Galeotti et al. 1997revealed that cisapride prevented dicylomine-induced amnesia in micesuggesting it plays an important role in modulation of memory processes.No further studies have been published. 9 0.2222 LEUCINE Enkephalin.Meilandt et al. 2008 found that ENKEPHALIN enkephalin elevations maycontribute to cognitive impairments in mice models of AD. 10 0.2222MLN4924 An ubiquitin-like protein with roles relevant to cellularprocesses important for cancer cell survival. 11 0.22222-(trifluoromethyl)- 10H-phenothiazine 12 0.2222 brucine An alkaloidantagonist at glycine receptors and paralyzes inhibitory neurons. It isa low potency M₁ positive allosteric modulator. There is high expressionof M₁ in areas of the brain responsible for learning, cognition, andmemory. 13 0.2222 Clodronate A bisphosphonate that affects calciummetabolism and inhibits bone resorption. Park et al. 2017 concluded thatin mice studies clodronate diminishes brain perivascular macrophageswhich prohibits amyloid-beta from damaging brain blood vessels. However,this effect is limited to a few weeks. 14 0.1667 Vincristine sulfate Analkaloid that irreversibly binds to microtubules and spindle proteins.It is an antineoplastic agent used to treat a variety of cancers. 150.1667 AZ 10417808 A selective caspase-3 inhibitor. 16 0.1667 CCCP Aproton ionophore. 17 0.1667 Flurofamide A potent inhibitor of bacterialurease. 18 0.1667 Chelidonine (+) An inhibitor of tubulin polymerizationinducing a G2/M mitotic arrest. Dickey et al. 2006 reported thatchelidonine reduced tau levels in vitro. 19 0.1667 Commonly known asturmeric. It is a scavenger of oxygen species and inhibits lipidperoxidation as well as peroxide-induced DNA damage. Small et al. 2018found that daily oral curcumin may lead to improved memory and attentionin non- demented adults. Zhang et al. 2006 concluded that curcumin mayenhance amyloid-beta uptake by macrophages in AD patients. Lin et al.2008 reported that curcumin significantly blocks the formative effect ofiron on neurofibrillary tangles in vitro. Several studies have revealedanti-Alzheimer's effects in mice and rat models (Lim et al. 2001,Garcia-Alloza et al. 2007, Ahmed et al. 2011). 20 0.1667 rizatriptan Aselective agonist of serotonin type 1B and 1D receptors.

TABLE 5B2 Drugs Identified Using Gene Expression Panels of TopBiomarkers CFG ≥ 10 (n = 112 unique genes; 53 increased and 59decreased). Panel of genes increased in expression: LMNA, FOXO3, CCND2,PMP22, BCAM, ELOVL6, HFE, NAV2, SLC1A7, FTL, MAPT, GFAP, LDLR, C4A,SNCA, THRA, TREM2, CSF1, IL1A, NRP2, GAP43, RCOR1, KIDINS220, CHAT,NPTX2, PON2, ANK1, IGF1, IGHG1, KLF3, FXYD1, COX6A1 AXL, PER1, SH3RF2,PPP2R2B, CLDN10, RGS10, FCGR1A, ITGB5, APOA1, WASF2, ZBTB16, OPHN1,ARG2, SHC3, TSPAN5, NLGN3, ARSB, AIMP2, CSNK1A1, RPL38, BACE1 Panel ofgenes decreased in expression: GSK3B, APOE, HELZ, VEGFA, HSPA5, ZFP36L1TGFB1, NDUFA5, ITPKB, DKK1, NOCT, SLC44A1, RHEB, NKTR, PGK1, SALL3,WDR45, CSF1R ICAM1, ABCA7, INPP5D, GAPDH, DUSP6, SREBF1, UQCRC1, TPK1,GSTM3, MICA, DLD, PSMA4 PSEN1, GUSB, BST2, CD36, NDUFS3, CTSS, MPEG1,TYROBP, B2M, RNASET2, FNBP1, USPL1 CEP350, FDPS, MTF2, RAB7A, PTGS2,NPC2, LYST, SERTAD3, SEC24A, HSBP1, SNRK, TRIM38 NUP214, UBE2I, ASPHD2,UBE2L3, ZC3HAV1. Rank Score Drug Description 1 0.1038 Proparacainehydrochloride Local anesthetic 2 0.0943 BRD-K00944562 3 0.0943BRD-A80151636 4 0.0943 BRD-K05361803 5 0.0943 BRD-K82137294 6 0.0943BRD-K34206396 7 0.0943 Pioglitazone A drug of the thiazolidinedione(TZD) class with hypoglycemic (antihyperglycemic, antidiabetic) action,used to treat diabetes 8 0.0849 TENOXICAM NSAID 9 0.0849 AC-1133 100.0849 Vincamine An antihypertensive with vasodilatory effects. 110.0755 5-nonyloxytryptamine An 5-HT1B selective agonist. 12 0.0755CINANSERIN A serotonin antagonist. 13 0.0755 Phenoxazine A dye whichconsists of an oxazine fused to two benzene rings. 14 0.0755 elesclomolAn inducer of heat shock protein 70 that activates natural killer cell-mediated tumor killing. 15 0.0755 curcumin A scavenger of oxygen speciesand inhibits lipid peroxidation as well as peroxide-induced DNA damage.16 0.0755 TOLAZAMIDE A sulfonylurea with hypoglycemic activity. 170.0755 Gly-Gly-delta-N-(phosphonacetyl)-L- ornithine 18 0.0755 bestatinA metalloprotease inhibitor selective for aminopeptidase. 19 0.0755levofloxacin A fluoroquinolone antibiotic. 20 0.0755 valaciclovir A DNApolymerase inhibitor.

TABLE 5B3 Drugs Identified Using Gene Expression Panels of PredictiveBiomarkers in All (n = 31 genes; 14 increased and 17 decreased). Panelof genes increased in expression: FCGR1A, GAP43, MAPT, HFE, RGS10,CALHM1 ARSB, LOC101928760, LOC101928123, RAB7A, TYMSOS, LOC100499194,ITPKB, LOC105371414 Panel of genes decreased in expression: NDUFA5,SEC24A, PSMA4, UBE2L3, NPC2, GUSB, TGFB1, TRIM38, CD40, ZNF345, IGF1,LOC101927027, MIS18BP1, RHEB, CARD11, NKTR, MS4A14 Rank Score DrugDescription 1 0.1818 CUNEATIN METHYL ETHER 2 0.1818 GR 159897 A potentand selective NK₂ receptor antagonist. 3 0.1818 Compound 58 4 0.1818ROLIPRAM A selective phosphodiesterase-4 inhibitor. 5 0.1818BRD-K01089529 6 0.1818 BRD-K15888437 7 0.1818 BRD-K17025677 8 0.18187618107 9 0.1818 BL-074 10 0.1818 BRD-A79981887 11 0.1818 BRD-A3216416412 0.1818 BRD-K02562327 13 0.1818 BRD-K74767048 14 0.1364 vorinostat Ahistone deacetylase inhibitor. 15 0.1364 curcumin A scavenger of oxygenspecies and inhibits lipid peroxidation as well as peroxide-induced DNAdamage. 16 0.1364 trichostatin A A histone deacetylase inhibitor. 170.1364 JW-7-24-1 18 0.1364 geldanamycin A benzoquinone antineoplasticantibiotic isolated from the bacterium Streptomyces hygroscopicus. 190.1364 MAPP, L-erythro 20 0.1364 Piperacetazine An antipsychoticprodrug.

TABLE 5B4 Drugs Identified Using Gene Expression Panels of PredictiveBiomarkers in Males (n = 34 genes; 15 increased and 19 decreased). Panelof genes increased in expression: FCGR1A, GAP43, MAPT, KIDINS220, AIMP2,RGS10, PER1, RAB7A, KLF3, CALHM1, BACE1, ARSB, LOC101928123,LOC100499194, ITPKB Panel of genes decreased in expression: NDUFA5,SEC24A, PSMA4, UBE2L3, NPC2, BST2, TGFB1, TRIM38, ZNF345, IGF1, VEGFA,LOC101927027, MIS18BP1, RHEB CARD11, NKTR, MS4A14, B2M, EPB42 Rank ScoreDrug Description 1 0.1786 Triamcinolone A syntheticglucocorticorsteroid. 2 0.1786 N20C hydrochloride Non-competitive NMDAreceptor open-channel blocker. 3 0.1786 manumycin A An antibiotic thatacts as a potent and selective farnesyltransferase inhibitor. 4 0.1786NCGC00183397-01 5 0.1786 BRD-K71917235 6 0.1786 BRD-A32164164 7 0.1429L-690,330 8 0.1429 PERHEXILINE A carnitine CPT1 and CPT2 MALEATEinhibitor. 9 0.1429 Clobetasol propionate A corticosteroid. 10 0.1429 GR159897 A NK2 receptor antagonist. 11 0.1429 NOBILETIN An O-methylatedflavone that has the activity to rescue bulbectomy- induced memoryimpairment. 12 0.1429 ENDECAPHYLLIN A glucose tetra-(3- Xnitropropanoate) ester. 13 0.1429 Flurandrenolide A corticosteroid. 140.1429 SDZ WAG 994 A potent and selective A1 adenosine receptor agonist.15 0.1429 Timolol maleate salt A non-selective beta-adrenergicantagonist. 16 0.1429 RHAPONTIN A crystalline glucoside found inrhubarb. 17 0.1429 16759925 18 0.1429 simvastatin A HMG-CoA reductaseinhibitor. 19 0.1429 2541665-P2 20 0.1429 Compound 58

TABLE 5B5 Drugs Identified Using Gene Expression Panels of PredictiveBiomarkers in Females (n = 12 genes; 6 increased and 6 decreased). Panelof genes increased in expression: DEFB104B, LINC01398, CHAT, RTCB,LOC105371414, PER1 Panel of genes decreased in expression: ITPKB, GUSB,CD40, SERTAD3, TBRG4, MS4A14 Rank Score Drug Description 1 0.2857Fluticasone propionate A synthetic trifluorinated glucocorticoidreceptor agonist. 2 0.2857 Anisomycin An antibiotic isolated fromvarious Streptomyces species. 3 0.2857 DIGOXIN A cardiotonic glycosideobtained mainly from Digitalis lanata. 4 0.2857 NICARDIPINE A calciumchannel blockader HYDROCHLORIDE with vasodilatory properties. 5 0.2857BRD-K06593056 6 0.2857 Inhibitor BEC A competitive inhibitor ofhydrochloride arginases I and II that causes NO-dependent smooth musclerelaxation. 7 0.2857 Emetine A protein synthesis inhibitorDihydrochloride derived from ipecac root. Hydrate (74) 8 0.2857Importazole A nuclear transport receptor importin-beta inhibitor. 90.2857 Salermide An inhibitor of SIRT1 and SIRT2 causing tumor-specificapoptotic cell death. 10 0.2857 BRD-K72264770 11 0.2857 dibenzyline Analpha-adrenergic antagonist. 12 0.2857 CGP-60474 A cyclin-dependentkinase inhibitor. 13 0.2857 HG-5-88-01 14 0.2857 Scopolamin-N-oxide Anantagonist of the hydrobromide muscarinic acetylcholine receptor. 150.2857 REV-5901 An antagonist of cysteinyl- leukotriene receptors. 160.2857 TRANS-7-HYDROXY- A dopamine D3 receptor PIPAT ligand. 17 0.2857Biotin Vitamin B7. 18 0.2857 NNC 711 An anticonvulsant that works as aselective inhibitor of GABA uptake by GAT-1. 19 0.2857 L-693,403 maleateσ ligand selectivity over the dopamine D2 receptor. 20 0.2857 W-7hydrochloride Calmodulin antagonist.Table 5C. Drug Repurposing using Crowd Extracted Expression ofDifferential Signatures (CREED)

TABLE 5C1 Drugs Identified Using Gene Expression Signature of TopBiomarkers CFG ≥ 12 (n = 18 unique genes; 8 increased and 10 decreased).Signed Rank Name Jaccard Index Description 1 Lorazepam 0.00727 Abenzodiazepine. 2 Finasteride 0.00656 A 5-alpha reductase inhibitor. 3Bromhexine 0.00649 An expectorant/mucolytic agent. 4 Ethinylestradiol0.00641 A semisynthetic estrogen. 5 Dicumarol 0.00639 Isolated frommolding sweet- clover hay, with anticoagulant and vitamin K depletionactivities. 6 Letrozole 0.00613 A nonsteroidal inhibitor of aromatase. 7Promazine 0.0061 A phenothiazine derivative with antipsychotic andantiemetic properties. 8 Diisopropyl 0.00598 An irreversiblecholinesterase Fluorophosphate inhibitor. 9 Rapamycin 0.00568 A mTORInhibitor. 10 Doxorubicin 0.00549 A topoisomerase inhibitor. 11Artemisinin 0.00542 A sesquiterpene lactone obtained from Artemisiaannua, which has been recently found to have potent activity againstmany forms of malarial organisms. 12 Colchicine 0.0054 Microtubuleinhibitor. 13 Mifepristone 0.00526 Progestin antagonist. 14 Zopiclone0.00526 A central nervous system depressant and a sedative. 15Amlodipine 0.00526 Calcium channel blocker. 16 Busulfan 0.00524 Analkylating agent used in the treatment of CML. 17 Rosiglitazone 0.00524A selective agonist for PPAR GAMMA. 18 Norethindrone 0.00523 A syntheticprogestin. 19 Letrozole 0.00521 A nonsteroidal inhibitor of aromatase.20 Omeprazole 0.00517 Proton pump inhibitor.

TABLE 5C2 Drugs Identified Using Gene Expression Signature of TopBiomarkers CFG ≥ 10 (n = 112 unique genes; 68 increased and 64decreased). Signed Jaccard Rank Drug Index Description 1 Hydralazine0.01735 An antihypertensive. 2 Rofecoxib 0.0135 NSAID. 3 Ethylene Glycol0.0134 Dihydroxy alcohol. 4 Doxycycline 0.01339 A tetracyclineantibiotic. 5 Levamisole 0.0131 An anthelmintic drug that has been triedas an adjuvant to chemotherapy. 6 Suxamethonium Chloride 0.01301 Adepolarizing skeletal muscle relaxant. 7 Tiapride 0.013 A D2 and D3dopamine receptor antagonist. 8 Bupropion 0.01295 An antidepressant ofthe aminoketone class and a non-nicotine aid to smoking cessation. 9Promethazine 0.01295 A first generation antihistamine that is used anantiemetic. 10 Pyrazinamide 0.01278 A synthetic pyrazinoic acid amidederivative with bactericidal properties against Mycobacteriumtuberculosis. 11 Antimycin A 0.01277 An antibacterial that blockselectron transport between coenzyme Q and cytochrome c. 12 Metoprolol0.01273 Competitive beta-1 adrenergic receptor antagonist. 13 Catechol0.01271 It has a role as a genotoxin, an allelochemical and a plantmetabolite. 14 Azathioprine 0.01264 A purine analogue that is used as animmunosuppressive agent. 15 Gadopentetate 0.01262 A gadolinium-basedparamagnetic Dimeglumine contrast agent. 16 Epirubicin 0.01247 Ananthracycline topoisomerase inhibitor. 17 Propylene Glycol 0.01246 Usedas an organic solvent. 18 Thiabendazole 0.01239 A broad spectrumantihelmintic agent. 19 Leflunomide 0.01231 An immunomodulatory agent.20 Imatinib 0.01231 Tyrosine kinase receptor inhibitor.

For the top biomarkers (see, Table 5), all the evidence from discovery(up to 6 points), prioritization (up to 12 points), testing (state,trait—up to 6 points each if significantly predicts in all subjects, 4points if predicts by gender, 2 points if predicts in gender/diagnosis)were tabulated into a convergent functional evidence (CFE) score. Thetotal score could be up to 30 points: 18 from the experimental data and12 from literature data. The experimental data was weighed more than theliterature data.

Example 1

In this example, biomarkers for short-term memory were determined.

Longitudinal studies were conducted in psychiatric disorder subjects, apopulation enriched in memory retention abnormalities. The subjects hadblood gene expression data at multiple testing visits, and werephenotyped at each visit, including with Hopkins Verbal Learning Test(HVLT). Subject's electronic medical records were also available forlong term follow-up of outcomes.

In Step 1 Discovery, blood gene expression biomarkers were identifiedthat track memory using a powerful within-subject design in a cohort ofsubjects who displayed at least a 20% change in the retention measurebetween different visits (n=159 subjects, with 496 visits), normalized(Z-scored) across genders and various psychiatric diagnoses. In Step 2Prioritization, a Convergent Functional Genomics approach was used toprioritize the candidate biomarkers in Step 1, using publishedliterature evidence (genetic, gene expression and proteomic), from humanand animal model studies, for involvement in AD. In Step 3 Testing, anindependent cohort (n=127) from the one used for discovery was examinedfor whether the top biomarkers prioritized in Step 2 were predictive ofmemory retention measure (state), and of future positiveneuropsychological testing for MCI, AD or other dementia (trait), usingelectronic medical records follow-up data of the study subjects (up to12.81 years from initial visit).

The top biological pathways where the candidate biomarkers map wererelated to LXR/RXR activation, neuroinflammation signalingatherosclerosis signaling, and amyloid processing (Table 2).Co-directionality of expression data provide new mechanistic insightsthat are consistent with a compensatory/scarring scenario for observedbrain pathological changes. The STRING gene interaction analysis (FIG.2) revealed at least 3 networks. Network 1 (red) includes TREM2, alongwith GUSB and RHEB; it may be involved in reactivity and inflammatoryresponses. Network 2 (green) includes MAPT (tau), along with PSEN1 andSNCA; it may be involved in activity and cellular trophicity. Network 3(blue) includes APOE, along with TGFB1 and FOXO3; it may be involved inconnectivity and synaptic integrity. GSK3B is at the overlap of Networks2 and 3.

The top candidate biomarkers were prioritized for convergent evidencefor involvement in AD (Table 5). They also had prior evidence ofinvolvement in other psychiatric and related disorders, providing amolecular underpinning for the possible precursor effects of thesedisorders in AD.

Gene expression biomarkers that were predictive in independent cohortsof memory state and of future neuropsychological testing positive forcognitive decline were successfully identified. Top predictivebiomarkers for state were NKTR, ITPK, RGS10, PERI, and ARSB (FIG. 3A).The AUC ROCs ranged from over 0.7 for all subjects tested to over 0.8personalized by gender, and over 0.9 personalized by gender anddiagnosis. Top predictive biomarkers for trait were KLF3, CEP350, FOXO3,MAPT, and RHEB (FIG. 3B). The Cox Regression Odds Ratios ranged fromover 2-fold for all subjects tested to over 4-fold personalized bygender and diagnosis.

RHEB, which represents the best biomarker for male schizophrenia, wasidentified as a future Alzheimer Disorder Related Dementia predictor inmales with schizophrenia (FIG. 4). Subject Phchp098 was a male withschizophrenia (SZ) initially tested in 2009. The subject was firstdiagnosed with paranoid schizophrenia in 1977. In 2016, he was alsodiagnosed by neuropsychological testing with ADRD and impaireddecision-making capacity. At that time, he was 66 years old. Subject wasthe only subject so far withan ADRD diagnosis in the independentreplication follow-up cohort. We tested RHEB, the best predictivebiomarker for males with SZ (FIG. 2B). RHEB levels were Z-scored bygender and diagnosis. Subject Phchp098 had the highest levels of RHEB inthe lab testing visit compared to all the subjects with futureneuropsychological testing (FIG. 4A) and the highest level of RHEB fromall the 111 subjects in that cohort (FIG. 4B).

Based on the studies and analyses, the biomarkers with the top overallconvergent functional evidence (CFE) for relevance to memory and AD wereNPC2, TGFB1, ARSB, GUSB, and KLF3, and then GSK3B, MAPT (tau), APOE,PSEN1, and TREM2. The fact that key genes for AD brain pathology cameout of the unbiased whole-genome discovery was reassuring and served asde facto positive controls for the approach.

Some of the biomarkers are targets of existing drugs, such as lithium,antidepressants, and omega-3 fatty acids (FIG. 5; Table 3), of potentialutility in patient stratification and pharmacogenomics approaches.Moreover, the top biomarkers gene expression signature, uponbioinformatics drug repurposing analyses, yielded new drug candidates(such as pioglitazone and levonorgestrel), and natural compounds (suchas salsolidine, ginkgolide A and icariin). Thus, the signature can beused for targeted enrollment of patients in clinical trials for thesecompounds, which would increase the odds of success, and for objectivelymeasuring response to treatment.

The methods described herein provide a novel approach for discoveringbiomarkers of relevance to Alzheimer's disease, as well as testing thebiomarkers in independent cohorts. The results provide evidence forprecision medicine, diagnostics and therapeutics. The methods canprovide improved early diagnosis of risk and preventive treatment formemory disorders in general, and Alzheimer's disease in particular, thatresult in decreased quality and quantity of life, at a massive cost toindividuals, families and society.

In view of the above, it will be seen that the several advantages of thedisclosure are achieved and other advantageous results attained. Asvarious changes could be made in the above methods without departingfrom the scope of the disclosure, it is intended that all mattercontained in the above description and shown in the accompanyingdrawings shall be interpreted as illustrative and not in a limitingsense.

When introducing elements of the present disclosure or the variousversions, embodiment(s) or aspects thereof, the articles “a”, “an”,“the” and “said” are intended to mean that there are one or more of theelements. The terms “comprising”, “including” and “having” are intendedto be inclusive and mean that there may be additional elements otherthan the listed elements.

1. A method of mitigating and preventing memory dysfunction, Alzheimer'sdisease, and cognitive decline in a subject in need thereof, the methodcomprising administering a therapy to the subject, the therapy beingselected from the group consisting of one or more compounds from Tables5A1-A5, and 5B1-B5, and 5C1-C2, for which we claim new method of use. 2.The method of claim 1, wherein the therapy is a drug, a naturalcompound, and combinations thereof.
 3. The method of claim 2, whereinthe drug is, an antidepressant, pioglitazone, sulfadimidine, SB-203580,mesalazine, metamizole, levonorgestrel, meglumine, lymecycline,rimexolone, ketanserin, quipazine, cisapride, proparacaine, tenoxicam,bexarotene or lithium.
 4. The method of claim 2, wherein the naturalcompound is an omega-3 fatty acid, salsolidine, ginkgolide A, andicariin.
 5. The method of claim 4, wherein the omega-3 fatty acid isdocosahexaenoic acid.
 6. The method of claim 1, wherein the subject hasa psychiatric disorder.
 7. The method of claim 1, wherein the subject isa male subject.
 8. The method of claim 1, wherein the subject is afemale subject.
 9. A computer-implemented method for assessing a lowmemory state in a subject, and for assessing risk of future AlzheimerDisease and cognitive decline in a subject, the method comprising:computing a score based on RNA level, protein level, DNA methylation, asingle nucleotide polymorphism, for a panel of biomarkers chosen fromTable 2, Table 4, or Table 5 in a sample obtained from a subject;computing a score based on a reference expression level of the panel ofbiomarkers; and identifying a difference between the score in the sampleobtained from the subject and the score in the reference sample, whereinthe difference in the score in the sample obtained from the subject andthe score in the reference sample indicates a low memory state in thesubject, and a risk of future Alzheimer Disease.
 10. The method of claim9, wherein upon identifying a difference between the score in the sampleobtained from the subject and the score in the reference sample, themethod further comprises administering a treatment to the subject,wherein the treatment reduces the difference between the score in thesample from the subject and the score in the reference sample, in orderto mitigate the low memory state in the subject, and the risk for futureAlzheimer's Disease or cognitive decline in the subject.
 11. The methodof claim 9, further comprising administering a treatment.
 12. The methodof claim 11, further comprising measuring response to treatment usingthe change in score.
 13. The method of claim 11, wherein the treatmentis selected from lifestyle modification and administering a therapy. 14.The method of claim 13, wherein the therapy is selected by acomputer-implemented method selected from the group consisting of one ormore psychiatric compounds from Table 3, and wherein each therapyselection is based on a panel of one or more individual biomarkers. 15.The method of claim 11, wherein the therapy is selected based on a panelof individual biomarkers changed in a subject, by a computer-implementedmethod for therapy selection, and consists of one or more new compoundsin Tables 5A1-A5, 5B1-B5, and 5C1-C2.
 16. A method for assessing andmitigating memory dysfunction, Alzheimer's disease, and cognitivedecline in a subject in need thereof, comprising determining anexpression level of a panel of biomarkers listed in Table 2, Table 4, orTable 5 in a sample; wherein the expression level of the biomarkers inthe sample is different relative to a reference expression level;identifying the subject currently having or at risk of having futurememory dysfunction, Alzheimer's disease, and cognitive decline based ona biomarker panel score relative to a biomarker panel score of areference; and administering to the subject a therapy being selectedbased on the score from the group consisting of one or more compoundsfrom Tables 5A1-A5, and 5B1-B5, and 5C1-C2.
 17. The method of claim 16,wherein the therapy is an antidepressant, pioglitazone, sulfadimidine,SB-203580, mesalazine, metamizole, levonorgestrel, meglumine,lymecycline, rimexolone, ketanserin, quipazine, cisapride, proparacaine,tenoxicam, bexarotene, salsolidine, ginkgolide A, icariin,docosahexaenoic acid, an omega-3 fatty acid, lithium or combinationsthereof.
 18. The method of claim 16, wherein the sample comprises aperipheral tissue, blood, saliva, cerebrospinal fluid (CSF), serum,urine, or stool.
 19. A composition comprising one or more compounds fromTables 5A1-A5, and 5B1-B5, and 5C1-C2 for use in a method for treatingmemory dysfunction, Alzheimer's disease, and cognitive decline.
 20. Thecomposition of claim 19, wherein the compound comprises, anantidepressant, pioglitazone, sulfadimidine, SB-203580, mesalazine,metamizole, levonorgestrel, meglumine, lymecycline, rimexolone,ketanserin, quipazine, cisapride, proparacaine, tenoxicam, bexarotene,salsolidine, ginkgolide A, icariin, docosahexaenoic acid, an omega-3fatty acid, lithium or combinations thereof.
 21. The composition ofclaim 19, wherein the compound comprises one or more of the compoundsfrom Tables 5A1-A5, and 5B1-B5, and 5C1-C2.